Poulter fired an 11-under-par 61 Thursday to shatter a 34-year-old course record before persistent thunderstorms cancelled play Friday, reducing the tournament to 54 holes.
Saturday, Poulter carded a 5-under-par 67 to maintain the lead by just one stroke over Scotland's Paul Lawrie. Local favorite Emanuele Canonica is two shots behind Lawrie.
Poulter, the 2000 champion, used a 4-iron for his ace on the 200-yard 16th hole. He had a hole-in-one here last year and another at the Heineken Classic at the start of the 2001 season.
"I hit a 4-iron, straight at the stick and as soon as I hit it I knew it was going to have a reasonable chance," Poulter said. "It pitched seven feet short, checked up and dropped in. It's my third hole-in-one and I've won nothing for any of them."
Poulter added four birdies with just one dropped shot on the par-3 No. 2.
Lawrie had nine birdies and signed for a 63 on Saturday.
Morning fog at Olgiata Golf Club delayed play by 40 minutes on Saturday and six golfers were unable to finish their rounds.
Needing to make up a 10-shot deficit, Lawrie produced a brilliant round to move within striking distance. But the 1999 British Open winner missed a chance to take the lead when a four-foot putt on the par-5 15th for a birdie lipped out and another birdie opportunity on the 17th from eight feet also failed to drop.
"It's not great when you stand on the first tee and you are 10 behind, especially after a good first day," Lawrie said. "But I got a good score and gave myself a chance for tomorrow."
Trying to become the first Italian to win here since Massimo Mannelli in 1980, Canonica fired a 65 to bring him to 13-under par.
"Three shots is a lot but not impossible and I feel confident I can catch Poulter even if with two rounds still to go it would have been easier," Canonica said. "I don't feel too much pressure for being an Italian. Sure, it would be lovely to be the first Italian winning the Italian Open after so many years."
Australian Jarrod Moseley is four shots out after a second straight 66. Henrik Nystrom of Sweden and Angel Cabrera of Argentina sit at 11-under-par.
The cut fell at 3-under par with 70 professionals and one amateur moving into the final round.