On the fourth day, the Victoria native used a late move to grab the pink jersey back from the Spanish rider.
Hesjedal caught everyone by surprise Saturday when he broke away from the peloton with just over a kilometre to go. Rodriguez tried to follow him, but in the end, he was forced to give Hesjedal back the top spot.
"Today was really hard," said Hesjedal, who earlier in the race became the first Canadian to take the overall lead. "But as I was feeling fine at the end, I wanted to test myself, even if I risked going out. Luckily, it worked.
"In the next few days I'll try to keep the jersey, which I'm happy to have taken back ... My teammates and I will always try to give everything."
On the first big summit finish of the race as it entered the Alps, Andrey Amador won the 14th stage in five hours 33 minutes 36 seconds.
He edged out Jan Barta and Alessandro De Marchi on the 206-kilometre trek from Cherasco to Cervinia.
Rodriguez said he will try to take the jersey back on Sunday.
"I knew today would be a hard stage," Rodriguez said. "I prefer shorter and steeper hills, and the weather was really cold ... I tried to attack in order to defend the pink jersey but we didn't manage."
Dominique Rollin of Boucherville, Que., finished the stage 93rd to end up 131st overall. Christian Meier of Sussex, N.B., crossed the line in 138th to move to 144th overall. Svein Tuft of Langley, B.C., finished 144th and is 163rd overall.
The 14th stage was mostly flat for the first 135 km. The route ascended over the next 50 km, with two Category 1 climbs.
The break came a third of the way into the stage, when Barta, Amador, De Marchi, Nelson Oliveira, Matteo Montaguti, Nikolas Maes, Pierpaolo De Negri, Olivier Kaisen swiftly established a six-minute lead. Their advantage increased to 13 minutes.
With 65 km to go, Barta made a lone break and even held a 30-second lead over other in the breakaway group, until he was caught by Amador on the descent at Col de Joux.
"I am really proud and happy, I worked a lot to get in form for this Giro, I wanted to win a stage and I had tried other times. After so many days in the breaks I was scared I wouldn't do it," Amador said.
The Costa Rican was then caught on the 27-km ascent to Cervinia by De Marchi and Barta, their lead over the peloton dropping, although they managed to hold off the group.
Sprinters Matt Goss, Mark Renshaw, Brett Lancaster and Juan Jose Haedo all withdrew from the Giro before Saturday's race. Mark Cavendish remains in the race and now has a 42-point lead in the red jersey standings as Goss had been his closest rival.
Monday is a rest day. The Giro ends on May 27 in Milan.