Hamish Marshall smashed 102 not out off 97 balls to help Gloucestershire beat Middlesex by three wickets with eight balls remaining at Bristol.
After winning the toss, Middlesex lost England duo Ed Joyce and Andrew Strauss cheaply, but Eoin Morgan's 82 helped them post 183-9 from their 40 overs.
Gloucestershire then lost regular wickets as Marshall lacked support.
But the New Zealander found a useful ally in recent signing Richard Dawson (17 not out) as they edged home.
Former England off-spinner Dawson, batting at number nine, hit a straight six off Tim Murtagh as he and Marshall added 33 for the eighth wicket.
As in their Twenty20 Cup-winning success, Middlesex were indebted to spinners Shaun Udal (3-36) and Murali Kartik (2-22) for putting the brakes on the hosts in mid-innings.
But crucially, Marshall - one of only four Gloucestershire players to reach double figures - was dropped by Steve Finn off Udal's bowling when he had only 46.
Earlier, seamer Steve Kirby took 4-27 for the home side, who also gave a debut to Tasmanian pace bowler Brendan Drew, who has signed a short-term deal to replace fellow Australian Marcus North as overseas player.
North has returned down under after being called up for Australia A.
Hamish Marshall told the BBC:
"Steve Kirby and Jon Lewis bowled well and put them under pressure by taking early wickets.
"When you are chasing, you have to be positive but not reckless. The boys are pretty happy but there are still two big games to go."
"Batting with Mark [Hardinges] we got ourselves into a position where we were well set up."
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