
The pair, who had never met on opposite sides of the court, were the last two Australians remaining in the field.
Hewitt let his temper rip as he complained about what he considered dubious line-calling at an event missing the electronic Hawkeye system used at major tennis events.
The Aussie felt hard done by in what he called "pretty ordinary" umpiring.
"What's going on with all the refs these days? They're useless. Central umpires have gone into the shells since Hawk-Eye's come in. In smaller tournaments they've still got to stand up and make decisions," said Hewitt.
"One week they've got Hawk-Eye and the next week they don't."
The loss in just over 90 minutes leaves Hewitt with only three matches on his ledger going into Monday's start of the Australian Open.
Guccione's win helped him start clawing back points after an early loss last week in Adelaide, where he lost the final a year ago.
Left-hander Guccione will next face Czech fourth seed Tomas Berdych in the last eight.
The loss was a huge blow to Hewitt, the former number one who now stands 24-6 against countrymen.
But he paid his due to Guccione.
"I'm more frustrated because I felt I played a pretty good match out there," he said. "I feel I was the better player all match and I think the stats show that.
"You've got to take your hat off to him. In the big points he served well."
Since the start of the century, Hewitt has gone 13-1 against compatriots, his lone loss against to Patrick Rafter in Cincinnati, 2001. Hewitt was denied the Sydney quarter-finals for the seventh straight time.
French men suffered mixed results, with top seed Richard Gasquet crashing out to Russian Dmitry Tursunov, 3-6, 6-3, 6-4.
But veterans Sebastien Grosjean and Fabrice Santoro sailed through. Grosjean beat eight seed Fernando Verdasco of Spain 6-3, 6-4 while racket magician Santoro dazzled Belgian Steve Darcis 6-1, 6-3.
Women's seeds swept through, led by world number one Justine Henin, who played her first match of the season after a walkover to crush Kaia Kanepi 6-2, 6-0 for a place in the quarter-finals.
Russian Svetlana Kuznetsova was equally ruthless with Italy's Francesca Schiavone, 6-4, 6-0. French Open finalist Ana Ivanovic, the number four, beat Slovenian Katarina Srebotnik 6-3, 3-6, 6-2.