"We want to stay in the EU, but change a number of treaties that are hurting our economy, our businesses," the nattily-dressed 31-year old said on his first day as chief of the protest party.
Champagne toasts over, Di Maio is rolling up his sleeves for the M5S's biggest challenge: snatching power from the centre-left in the general election next spring.
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The movement has set its sights on gold after months spent neck-and-neck with the Democratic Party in surveys. Although an Italian "Brexit" is not among its electoral promises, the M5S has long called for a referendum on the euro and recently signalled that a possible exit from the single currency could become a central issue.
"We have presented a seven-point programme to the European parliament on the euro, with a referendum on the single currency as the final point," Di Maio said.
"If the attitude is one of openness, we are willing to take part in a discussion on changing the rules of the game.
M5S co-founder Beppe Grillo congratulates Di Maio on his win. Photo: AFP
"If it isn't - and only in that case - we would ask the Italians as a last resort to give their opinion on the euro in a consultative referendum," he said.
M5S, which bases much of its appeal on fighting cronyism and corruption, has always defined itself as on neither the political left nor right, and presents voters with a hotchpotch of policies from across the ideological spectrum.
Critics have accused it of flip-flopping on key foreign policy issues, swinging for example from being intensely anti-Vladimir Putin at its founding in 2009 to supporting the Russian leader.
While the movement expresses a distrust of traditional media outlets like US President Donald Trump, Di Miao said an Italy run by M5S would not immediately ally with the American leader.
"On issues like North Korea, we share the US's apprehension," he said. But the movement refuses to bow to Trump's demand that European countries spend more on the North Atlantic Treaty Organization (NATO).
Luigi Di Maio. Photo: AFP
"We are certainly not willing to refinance the NATO programme with an extra 14 billion euros," Di Maio said, referring to the amount he said would be needed for Italy to raise its spending to roughly two percent.
"We have always said that, and we will tell Donald Trump".
Though the M5S was driven at the start by largely leftist ideals such as calls for a minimum wage, its leaders have moved across the spectrum since, often echoing the anti-immigration Northern League party.
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Di Maio in particular has been accused of attacking non-governmental organizations that save migrants' lives. Hundreds of thousands of people have set off from the coasts of north Africa for Italy by boat over the past few years, making immigration the hottest topic on the table at the elections.
The departures dropped sharply after an alleged "agreement under the table" in July between Italian officials and major traffickers which saw at least one well-known smuggler baron tasked with stopping smuggling.
"The government's is a mere containment strategy, it's not going to last in the long term," Di Maio said.
"We had proposed the EU make it possible for people to apply to come to the EU from their departure countries.
"That way we could have filtered the requests at the point of origin between those who have the right to come, as political refugees, and those who don't because they are economic migrants," he said.
Instead, the M5S was "very worried", he said, about "the government making deals in Libya with dubious organisations that violate human rights".
By Ljubomir Milasin