The thieves bypassed alarm and security systems on Saturday evening at the MuMe museum in Messina, making off with three of the five surviving panels of the San Gregorio Polyptych, dated to 1473.
They also took the double-sided panel depicting the Virgin Mary and the dead Christ in Pietà, which was removed from an armoured display case, the news agency LaPresse reported.
They removed all five panels of the San Gregorio Polyptych from their frame, but abandoned two behind a wall while fleeing.
Art expert Alberto Fiz estimated the value of the stolen works at 70 million euros ($A115 million) to 80 million euros ($A131 million).
"We are devastated by what happened. They were two of Antonello da Messina's most important and best-known works. It is a tremendous loss for the museum, the city, the community and the art world," MuMe director Marisa Mercurio told the ANSA news agency.
Mercurio said the theft occurred just before 10pm local time, and that the alarms were working and the guards were on site. She described the works as central to the region's heritage.
"These are not merely works by Antonello da Messina," she told Sky TG24.
"They are works that hold considerable significance in terms of our identity, as well as, of course, their artistic and cultural value, because they are works by our very own Antonello da Messina."
Messina's top culture official, Enzo Caruso, called the theft "more than a loss" and "a disaster".
"Messina without Antonello is deprived of the very essence of the greatest artist of the European Renaissance," Caruso told Sky, calling for the swift recovery of the stolen works.
Caruso noted that international interest in the artist had intensified since Italy's Culture Ministry acquired Antonello's devotional painting Ecce Homo for $US14.9 million ($A21.1 million) through negotiations with Sotheby's in New York earlier this year.
Lynda Albertson, an art-crime analyst, said the works are instantly recognisable, impeding any effort to sell them through reputable channels.
"Stealing an Antonello may be far easier than selling one. If these are ordinary thieves, they will soon discover that — to their cost," Albertson, who is the chief executive of the ARCA art crimes research association, told the Italian daily La Repubblica.
She said such famous artworks often pass from one criminal to another, serving as collateral for other illicit activity.
The museum was closed on Sunday while investigators gathered evidence. Neither museum officials nor the Carabinieri could immediately be reached for comment.
The Ferragosto holiday, which marks the feast of the Assumption of Mary, marks the height of the Italian summer holiday season, and was being celebrated in Messina with a procession that culminated in prayers and a blessing in the main Piazza Duomo around the time of the heist.
The theft comes just days after police in the northern Italian city of Parma announced that they had recovered three stolen artworks by Renoir, Cézanne and Matisse worth about 10 million euros ($A16 million).
Five people have been detained in the March 22-23 theft from the Magnani Rocca Foundation located in the Parma province.
