With a superb climb of the Passo dello Stelvio De Gendt finished the so-called queen stage of the Giro in six hours 54 minutes and 41 seconds.
Victoria's Ryder Hesjedal remained second overall after finishing sixth in Saturday's stage 3:36 behind De Gendt. He is 31 seconds behind Rodriguez heading into the final stage.
Saturdays stage was the toughest of this year's race, with the 219-kilometre trek from Caldes culminating in a 22.5-kilometre ascent up the snow-covered Stelvio, which has a maximum gradient of 12 per cent.
The route also included the legendary Mortirolo climb, described by the seven-time Tour De France winner Lance Armstrong as the hardest climb he's ever ridden.
The Giro finishes on Sunday in Milan after a 30-kilometre individual time trial.