Stybar (Etixx-QuickStep) timed his attack to perfection to win the 207km stage between Camaiore and Pomarance stage and move into an overall lead of nine seconds.
The Czech rider went clear at the top of the tricky final climb, opening up a gap on the short descent and holding off a determined chase by the back markers.
"I knew about the finale since I recce'd it with our sport director Davide Bramati," Stybar said. "I knew it suited me perfectly. I also knew that I had good form since I did well at the Strade Bianche [2nd].
"I attacked at the right moment when it was very steep just inside three kilometres to go. I'm delighted to lead the race now, I'll try to win the overall but I can't promise I'll do it because there's an uphill finish on Sunday that might be too hard for me".
Peter Sagan (Tinkoff) was second, leading home an elite group of chasers including Edvald Boasson Hagen (Dimension Data) and Australia's Simon Clarke (Cannondale).
Local favourite Diego Ulissi attacked on the slopes of Il Cerreto but he was reeled in and passed by Stybar near the summit.
Vincenzo Nibali (Astana) gave chase in the final kilometre but was unable to bridge the gap and the Italian was swallowed up by the peloton in the sprint to the line.
American squad BMC Racing won the opening team trial stage on Wednesday to put Daniel Oss into the leader's blue jersey but the 10-second stage winner's time bonus saw Stybar take over the overall lead.
The Etixx-Quick-Step rider has an advantage of nine seconds over Oss' teammates, Greg Van Avermaet and Tejay van Garderen.
The seven-day Tirreno-Adriatico continues with a 176km ride from Castelnuovo Val di Cecina to Montalto di Castro.