Pauli won thanks to a brace from Dapo Afolayan and a goal from the club's top scorer Marcel Hartel and return to the top division for the first time since being relegated in 2011.
Pauli were promoted alongside fellow northern side Holstein Kiel, who secured their first ever top-division season with a home draw against third-placed Fortuna Duesseldorf on Saturday.
Former Bolton and West Ham striker Afolayan gave the home side the lead after just seven minutes and doubled up early in the second-half with a tap in at the far post.
Hartel headed in a third with 22 minutes remaining, his 17th league goal of the season.
Osnabrueck's Lars Kehl scored a penalty in stoppage time but the celebrations began shortly after at St Pauli's Millerntor Stadium, just down from the city's notorious Reeperbahn.
Known for their skull and crossbones flag and identification with left-wing causes, Pauli have an international reputation which is larger than their sporting achievements.
The promotion continues Pauli's stunning rise under 31-year-old America-born coach Fabian Huerzler.
Huerzler took over just before Christmas in 2022 with the club hovering dangerously close to relegation. He piloted a rapid rise up the table to fourth at season's end, narrowly missing promotion last season, but getting the job done the second time around.
Pauli are first with one match remaining and will be champions of the second division by matching second-placed Kiel's result next weekend.
Duesseldorf are guaranteed to finish third and will play a two-legged playoff against the third-last side in the top division, currently Union Berlin.
Pauli's promotion, the sixth in club history, is even sweeter given city rivals Hamburg SV again missed out on returning to the top division.
Hamburg, a one-time Champions League winner, were relegated for the first time in 2018-19 but have failed to return to the top division in the six seasons since.
Hamburg, currently in fourth spot, have finished fourth three times and third twice, losing the relegation playoff on both occasions.
