Bradley Wright Phillips struck a goal in each half to put Saints in control and, although Jimmy Kebe pulled one back for Reading, the home side were unable to mount a fightback.
It was a deserved win for a young Southampton side, although the game could have been very different had Jack Cork not chested Kevin Doyle's second minute shot off his own line.
A Reading goal then would have been a case of business as usual for a side which had dropped just two points at home all season and boasted three successive wins to maintain their impressive push for promotion.
But Jan Poortvliet's young Saints side had clearly not read the script.
Morgan Schneiderlin saw a low shot blocked and Adam Lallana forced a low save from Marcus Hahnemann as Southampton's neat build-up play began to carve out chances.
They went even closer on 10 minutes when Schneiderlin helped the ball forward and Wright Phillips saw a clear overhead kick fly just wide.
But the visitors were deservedly in front on 14 minutes when Lloyd James' deep cross from the right tempted Hahnemann to start to come but stop, allowing Wright Phillips to cleverly steer a header into the far corner.
It could have been worse for the home side as Saints had two penalty appeals waved away.
Hahnemann then made an outstanding reaction save to keep out Lallana's point-blank header, but Saints were 2-0 up four minutes into the second half as Wright Phillips easily cut inside Liam Rosenior and rifled a shot beyond the surprised keeper.
That prompted Steve Coppell to bring Czech international midfielder Marek Matejovsky off the bench and he had an immediate impact, starting the move which led to Reading's goal.
Doyle's resulting shot from Chris Armstrong's cross was well saved by Kelvin Davis, but the ball dropped for Kebe to rifle through a crowd of legs and secure a lifeline for Reading.
Reading fans thought they were celebrating an equaliser when James Harper dived to head home Doyle's cross, but the Reading captain was clearly offside.
Southampton's hard-working youngsters were hanging on though and their tired legs were exposed two minutes from time when Bryn Gunnarsson's cross found Doyle in space only for the striker to head over with the goal at his mercy.