The M5S rules in coalition with Matteo Salvini's nationalist League, which won a resounding victory in Italy on the back of an "Italians First" campaign, dealing a blow to the Movement and threatening the stability of the government.
"You decide. I am asking to put my role as party leader to the vote," Di Maio said on the M5S blog on Wednesday. "If the Movement renews its faith in me, we'll get to work... with even more commitment and dedication," he added.
The vote on whether he should stay on will take place on the M5S online platform on Thursday.
The EU vote results confirmed the reversal of fortunes of the ruling parties, with M5S -- which pocketed 32.5 percent at the general election -- taking home just 17 percent on Sunday compared to the League's 34 percent.
"I had promised to get the Movement into government... and we did. I never gave any less than 100 percent," Di Maio said.
"I would never have thought working hard would be a fault," he added, in an apparent reference to Salvini, who famously spent fewer than 20 days in his office between the start of the year and the vote as he toured the country campaigning.
The M5S chief has been criticised by part of the Movement's base for taking on too much with his three roles as party head, deputy prime minister and economic development, labour and social policies minister.
Di Maio has blamed the M5S's poor performance on low voter turnout, as well as a mud-slinging campaign by the League against which it was slow to retaliate.
He received a show of support Wednesday from comedian Beppe Grillo, the M5S co-founder, who remains an influential figure within the Movement despite withdrawing from the political scene to focus on his stand-up career.
Beppe Grillo (L) and the party's new leader Luigi Di Maio with the Five Star Movement's new logo. Photo: Andreas Solaro/AFP
"The Movement has suffered a defeat and must react," he admitted on his website. However, he said he was "wounded" by those M5S members who were acting "as if it were a drop in the sales of a multinational company".
"Luigi has not committed any crime, he is not involved in any scandal. He must carry on the fight," Grillo said.