Barnsley substitute Martin Devaney headed home an injury-time winner from close range. Two free-kicks from Grant McCann had given the Tykes a two-goal lead before Marek Saganowski scored from close-range just before half-time.
Barnsley were reduced to 10-men when Dominik Werling was sent off for a second bookable offence.
Saints Inigo Idiakez thought he had grabbed a point from 12 yards in injury time until Devaney's late effort.
Southampton boss George Burley:
"I thought we deserved to equalise but then we threw it away by being totally unprofessional. "It was an amazing second half. When they went down to 10 men I thought we had it in us to go on and win.
"We have an experienced back four and as a manager you have to be upset to lose like that."
Barnsley manager Simon Davey:
"Grant McCann's goals were world class free-kicks and I don't think anybody could have stopped them. "We were heavily beaten here last season but this is almost a new team so it made little difference.
"Domonik Werling cannot complain about being sent off. I hope he learns his lesson."
Southampton: Davis, Wright (Idiakez 81), Thomas, Dailly, Vignal, Hammill, Viafara, Safri, Surman, Saganowski (Rasiak 69), Wright-Phillips (John 46).
Subs Not Used: Bialkowski, Makin.
Booked: Safri.
Goals: Saganowski 45, Idiakez 90.
Barnsley: Muller, Nyatanga, Souza, Foster, Werling, Campbell-Ryce (Devaney 74), De Silva (Reid 90), Howard, McCann, Ferenczi (Togwell 66), Odejayi.
Subs Not Used: Colgan, Mostto.
Sent Off: Werling (70).
Booked: Werling, Howard, Campbell-Ryce, De Silva, Devaney.
Goals: McCann 27, 40, Devaney 90.
Att: 19,151.
Ref: Michael Jones (Cheshire).
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