US Immigration and Customs Enforcement gave the painting, entitled "Christ Carrying the Cross Dragged by a Rascal" by Girolamo Romano (1484-1566), to Corinne Hershkovitch, a French lawyer representing the descendants of the Gentili family.
The lawyer accepted the painting during a ceremony in Tallahassee, Florida.
"Thanks to the tireless efforts of those involved, we are now righting a wrong perpetrated more than 70 years ago," said Susan McCormick, the federal agent who investigated the case.
Lionel Salem, an heir to the Gentili estate, thanked US officials for their "fantastic work."
"The Gentili heirs are unanimous in wishing to convey the full extent of their immense gratitude," he said in a statement.
The painting has been displayed since March 18, 2011 at the Mary Brogan Museum of Art and Science in Tallahassee, where it was on loan from Italy's Pinacoteca di Brera art gallery in Milan, along with 50 other paintings.
The painting, which dates to around 1538 from an artist also known as Il Romanino, depicts Christ, crowned with thorns and wearing a copper-colored silk robe, carrying the cross on his right shoulder while being dragged with a rope by a soldier.
The exhibition displayed Baroque art from the Lombardy region of Italy.
Before the exhibition ended September 4, US Attorney Pamela Marsh became suspicious about the origins of the painting.
An investigation traced it to the Gentili family, who were Italian refugees living in France during the Vichy regime that collaborated with the Axis powers during World War II.
The family's art collection was sold in 1941 during a forced sale.
US authorities seized the painting last year for their investigation.
The Gentilis' grandchildren have sought for years through courts to recover their family's stolen artwork. They succeeded in 1999 in having five paintings displayed in the Louvre Museum in Paris returned to them.
According to the Tallahassee Democrat newspaper, which revealed the case in September, the painting dates from around 1538 and was insured for $2.5 million.
It was purchased by the Italian museum in 1998.
Similar cases are common in Europe and the United States after thousands of stolen artworks were sold by the Nazis or their collaborators.