Hesjedal was only six seconds behind in sixth place, but Rodriguez moved ahead of him thanks to a 20-second time bonus for the stage victory.
"Hats off to Purito," Hesjedal said in an email to The Canadian Press, referring to Rodriguez' nickname. "His team was incredibly strong and they took the initiative riding all day. He is the best in the world on that type of finish.
"It was an honour to wear the maglia rosa," the Victoria native added. "I can't be disappointed losing the jersey in those circumstances and I am pleased with my condition and will look to the rest of the race with confidence."
Hesjedal was the first Canadian ever to lead the race.
Rodriguez is 17 seconds ahead of Hesjedal in the overall standings, with Italy's Paolo Tiralongo 32 seconds back.
The Katusha rider, one of the pre-race favourites, finished the 186-kilometre leg from Civitavecchia to Assisi in four hours 25 minutes five seconds for his first Giro stage win.
"The goal is the podium and to fight for the Giro victory," Rodriguez said. "The last week will be super tough and you've got to race intelligently. ... No matter what happens, at least now I've worn the only leader's jersey missing from my collection in the three major races (with the Tour de France and Spanish Vuelta)."
Bartoz Huzarski of Poland was two seconds back in the stage. Italy's Giovanni Visconti finished third, four seconds behind.
The stage began at the port of Civitavecchia on Italy's western coast, then crossed into Tuscany with a difficult finish in the ancient hill town of Assisi.
With four kilometres to go, there was a tough climb to San Damiano with gradients as steep as 15 per cent. Then after a brief downhill section, the stage ended in Assisi's historical centre.
Dominique Rollin of Boucherville, Que., finished 74th to move up seven spots to 143rd overall.
Christian Meier of Sussex, N.B., was 159th to drop one spot to 147th, while Svein Tuft of Langley, B.C., finished 160th to fall one spot to 164th.
Frank Schleck, another pre-race favourite, struggled and finished 26 seconds behind Rodriguez to drop out of the top 10.
Filippo Pozzato withdrew before the stage after breaking a bone in his right hand in a fall at the end of Stage 9. Pozzato accepted responsibility for causing the crash, which resulted in a multi-rider pileup.
Stage 11 Wednesday is the race's longest, a 255-kilometre leg from Assisi to Montecatini Terme.
The race ends May 27 in Milan.
With files from The Canadian Press