The Dutchwoman covered the 22.5km route in 32 minutes and 52 seconds, one minute and 52 seconds clear of Lizzie Deignan (Boels-Dolmans).
Following Deignan's wheel into the Stade Velodrome in Marseille to a goosebump inducing crowd was Italian rider Elisa Longo Borghini (Wiggle High 5).
Amanda Spratt (Orica Scott) and Shara Gillow (FDJ Nouvelle Aquitane) were the top Australian finishers in fifth and sixth.
Van Vleuten started the stage as she finished on the Col d'Izoard - 43 seconds ahead of Deignan. With the reverse handicap rewarding rather than hobbling the best finisher, there were only two ways riders behind could race the stage - riding it like a time-trial and hope for the best, or wait for allies behind.
But as current Dutch national time trial champion Van Vleuten is hardly shabby racing against the clock, Deignan opted for collaboration, sitting up immediately once out of the stadium to join team mate Megan Guarnier and Longo Borghini.
Van Vleuten's lead quickly grew to one minute and 43 seconds after only several kilometres raced. The gap reduced to one minute and 36 seconds as Van Vleuten hit the only climb on the course and 7.5 kilometres left to race.
But it had extended back out to one minute and fifty seconds, only two kilometres from the finish. Van Vleuten was never going to lose this unofficial race against the clock.
"I didn't have the information it made it a bit hard so I thought just go and stay to my tactics," Van Vleuten said.
"But I saw out on the way (Megan Guarnier), Lizzie Armitstead (Deignan) and Longo Borghini) riding together the three of them and I think 'this may be hard.' So it made me a bit nervous.
"But I thought, stick to my plan, go as hard as possible and smash the climb and then I finally got the information I was one minute 40 and that made me happy.
"Izoard was really special to win on that uphill finish and I still get goosebumps if I think about it and this ambience is wonderful."