The Dutchman, one of the favourites to win the three-week race, hit the ground in a massive pile-up about six km from the line in a 235-km ride from Orbetello won by Ecuador's Richard Carapaz.
Slovenian Primoz Roglic retained the overall leader's pink jersey as the crash involving Dumoulin held up several overall contenders.
Team Sunweb rider Dumoulin, who started the day in fifth place in the general classification, crossed the finish line surrounded by his team mates, with blood dripping from his left knee.
"X-ray reveals no fracture for Tom Dumoulin," his team said in a statement.
Dumoulin, however, was unsure if he would be at the start of Wednesday's fifth stage.
"There are no fractures which is good but my knee is very swollen," he said.
"I wasn’t able to push any power in the last few kilometres so I don’t know how I will be tomorrow; the GC (general classification) is for sure gone."
Among those held up were twice champion Vincenzo Nibali of Italy and Briton Simon Yates, who limited the damage in losing 16 seconds to Roglic.
MASSIVE CRASH
The massive crash appeared to be caused by Italian Salvatore Puccio of Team Ineos, who was the first rider to lose his balance and was just avoided by Nibali.
"Salvatore was caught in the crash and apparently he was the one who caused the crash and that happens sometimes, unfortunately," said Team Ineos sports director Nicolas Portal.
"It's been really technical the past couple of days, the final 30 or 40 kilometres, town after town after town and so we say, 'It's Italy'."
Team Jumbo-Visma's Roglic leads Yates by 35 seconds and Nibali by 39 going into Wednesday's fifth stage, a 140-km stretch between Frascati and Terracina.
While Dumoulin was struggling to pedal to reach the line, Carapaz was the strongest of a small group in an uphill finish, holding off sprint specialist Caleb Ewan of Australia and Italian Diego Ulissi.
Team Movistar rider Carapaz, fourth overall last year, had lost 45 seconds after a late mechanical problem on Monday.
“Yesterday was a bad day for me. Today I rode with a new mentality and set out to gain a few seconds," Carapaz told reporters.
"I knew there were fast riders with me. I made my effort 600 metres out and, well, it's very emotional for me."
(Reporting by Julien Pretot; Editing by Alison Williams/Toby Davis/Ken Ferris)
