Saints suffered late frustration at Kingsholm as Gerbrandt Grobler scored a last-gasp try to help seal a 14-12 Premiership Rugby Cup victory for Gloucester.
After going ahead for the second time early in the second half, Saints looked set to claim victory after defending so well for so long.
But the Gloucester pressure eventually paid off as the home players called for referee Greg Macdonald to go to the TMO, and Grobler's try was awarded.
Billy Twelvetrees finished the job with the simple conversion as Saints were left to rue a late loss, having inflicted one on Wasps just five days earlier.
There were also several injury concerns for Saints ahead of the return to Gallagher Premiership action next Saturday as Piers Francis, Mike Haywood and Luther Burrell were all forced off at Kingsholm.
Francis had started the scoring on the night as some nice combination play with George Furbank opened the door for the centre to touch down.
James Grayson sent the conversion just wide in the wind and he was soon off target with a penalty after Twelvetrees was caught offside.
Saints had dominated all the early possession and even when Gloucester did get an opening, it came from a loose pass.
Grayson tried to find a team-mate just inside his own half but Matt Banahan intercepted and raced away to score, hitting Saints with a sucker punch.
Macdonald did consult his TMO, with replays showing there was a knock-on from Twelvetrees in the build-up, but the try stood.
Twelvetrees rubbed salt in the wounds with the conversion and Gloucester had a lead that their lack of territory and possession had not merited.
Saints, who had earlier lost Francis to a head knock, then suffered a second injury blow as Haywood had to be helped from the field on his 200th appearance for the club.
And Paul Hill, who was making his first appearance of the season, was also heading off the field soon after, having been yellow carded for making a tackle after not retreating the necessary distance from a quick tap penalty.
Gloucester then had a huge chance to score as full-back Jason Woodward broke through but after the ball was recycled, Ollie Thorley knocked on with the line in sight.
Saints held out until half-time as Gloucester struggled to threaten further despite having the man advantage.
And the Cherry and Whites were made to pay just after the restart as Tom Collins and Cobus Reinach combined brilliantly for a Saints score.
Reinach gave the ball to Collins, who kicked ahead for Reinach to put his own boot to the ball and dive on it to score.
Grayson converted superbly to put his team 12-7 up before Hill returned from the sin bin.
Saints then had to get through plenty of defensive work as Gloucester tried to produce a response.
The home side were huffing and puffing but they looked like they lacked the ideas to blow the Saints door down.
The Kingsholm faithful were growing more and more frustrated as the minutes ticked by, with the fury reaching fever pitch when Gloucester tried to use crossfield kicks to gain ground in attack.
But the Cherry and Whites supporters were finally given a reason to cheer when Macdonald, who appeared to miss a knock-on at a scrum, awarded the try to Grobler after a concerted spell of pressure.
Twelvetrees held his nerve to slot the simple conversion and draw another roar from his team's fans as they celebrated a second success of the season against Saints.
Gloucester: Woodward (Hudson 72); Banahan, Trinder (Purdy 54), Twelvetrees, Thorley; Atkinson, Heinz (c) (Braley 54); Hohneck (Rapava Ruskin 46), Hanson (Walker 46), Balmain (Knight 60); Savage, Slater (Ackermann 44); Evans, Kriel (Grobler 54), Clarke.
Saints: Furbank; Collins, Dingwall, Francis (Burrell 16 (Kellaway 71)), Naiyaravoro; Grayson, Reinach (Mitchell 60); Davis (Waller 54), Haywood (Fish 23), Hill (Garside 68); Ribbans, Moon (Ratuniyarawa 58); Wood (c), Ludlam, Harrison (Gibson 58).
Referee: Greg Macdonald