"Your honour, I am not guilty," Gotovina said as each of the seven counts against him was read out on Monday.
He is accused of committing atrocities against ethnic Serbs in 1995 during a Croatian offensive on Serb-held territory.
After four years on the run, Gotovina was arrested last week in Spain's Canary Islands and transferred to the International Criminal Tribunal for the Former Yugoslavia (ICTY).
If convicted he could get a maximum sentence of life imprisonment. A date for his trial has not yet been set.
Gotovina's arrest and transfer to the tribunal sparked outrage with some in Croatia who see him as a national hero of the 1991-95 war of secession, which led to Croatia's independence from the Yugoslav federation.
In particular, the Croatian offensive known as Operation Storm that Gotovina is charged over, is regarded as decisive in ending the war.
During that operation, between 150,000 and 200,000 Serb civilians fled to neighbouring Bosnia and Serbia and at least 150 Serbs were killed by troops under Gotovina's command, according the ICTY indictment.
Over the weekend thousands of angry Croatian nationalists took to the streets to protest Gotovina's arrest, accusing their government of "treason".
The European Union and the United States hailed the arrest as the removal of a major obstacle to Croatia eventually joining the EU.
The bloc made Gotovina's arrest a key condition for stronger ties.
Pressure for more arrests
Gotovina’s capture increases pressure on Bosnia and Serbia to arrest the two other most wanted fugitives of the court: Bosnian Serb wartime leader Radovan Karadzic and his military commander Ratko Mladic.
The two are wanted on charges including genocide over the 43-month siege of Sarajevo and the 1995 Srebrenica massacre of an estimated 8,000 Muslim males, the single worst atrocity in Europe since World War II.
Both remain at large despite a US$5 million US offer for information leading to their arrests.
NATO has failed in repeated efforts to capture Mladic and Karadzic, who remain heroes to nationalist Serbs.
Karadzic is thought to spend his time in either Bosnia or across the border in Montenegro and Mladic is widely believed to be hiding in Serbia proper.
Raids in Bosnia
On Monday NATO troops raided the home of a supporter of Karadzic in Bosnia-Hercegovina in a bid to pressurise the former Bosnian Serb strongman's network.
Soldiers searched the Pale residence of Dragan Stajcic, thought to be a close associate of Karadzic.
The police forces of Montenegro also apparently stepped up their efforts to locate Karadzic by raiding the home of his brother.
But Bosnian Prime Minister Adnan Terzic has said local authorities believed that Karadzic and Mladic were both in Serbia-Montenegro.
"We can only encourage authorities in Serbia-Montenegro to hand over Karadzic and Mladic as soon as possible since they are there and not in Bosnia," Mr Terzic told reporters, adding the information came from Bosnian institutions dealing with war crimes.
Serbia also hopes to start EU membership talks but its failure to help capture Mladic and Karadzic has hampered its bid to join the bloc.
War crimes prosecutor Carla Del Ponte is due to submit her assessment of Serbia's cooperation with the tribunal this week.
14 jailed over Vukovar
Serbia on Monday convicted 14 former Serb militia members over the massacre of nearly 200 Croatian prisoners of war and wounded during the battle of Vukovar in November 1991.
A special Belgrade court found them guilty of carrying out the executions on a farm at the end of the three-month siege of Croatia's easternmost town by local Serb rebels backed by Yugoslav army troops.
Members of the Serb territorial defence rounded up victims from a hospital where they had sought shelter and took them to pits where they were shot by firing squads.
In the most significant war crimes judgment yet made by a Serbian court, the judge handed out prison sentences ranging from five to 20 years.
Three Serb commanders charged with orchestrating the massacre are currently on trial before the ICTY.
Serbia's special war crimes court was set up with approval from the Hague tribunal in 2003 to show the country was able to face up to its bloody past.
Milosevic trial adjourned
Meanwhile judges at the war crimes court in The Hague on Monday adjourned the trial of former Yugoslav president Slobodan Milosevic for six weeks to give him time to rest.
Milosevic asked for an extended break to allow him to be sent to Moscow for medical treatment, but the judges rejected that request.
Milosevic faces over 60 counts over war crimes and crimes against humanity over his role in the 1990s Balkan wars. For the bloody war in Bosnian which left over 200,000 dead he faces separate genocide charges.