Prime Minister Ehud Olmert has denounced the attack, which struck a school in the centre of Ashkelon but caused no casualties, as an escalation of "unprecedented gravity in the campaign of terror waged by Hamas".
The rocket landed on a school in the port city on the Mediterranean just south of Tel Aviv on Tuesday night, causing significant damage, the army said.
In a statement received by AFP, the armed wing of Hamas, the Ezzedine al-Qassam Brigades, claimed responsibility for firing the rocket.
Israeli missiles tore through the Palestinian Interior Ministry in Gaza hours after Hamas fired the rocket into Ashkelon.
The Israeli air strike wounded at least three people.
The Qassam Brigades had already threatened to resume attacks inside Israel after an 18-month truce, warning of a "sea of blood" should a massive military assault on Gaza, launched in a bid to secure the soldier's release, not cease.
Tuesday's attack was the first in central Ashkelon, according to the army.
Palestinian militants frequently fire homemade rockets, which are notoriously inaccurate and generally have a maximum range of around 12 kilometres, into southern Israel.