The Royals had gone into the game as the only team in the division with an unbeaten home record this season.
Both Southampton goals came from Bradley Wright-Phillips, the first in the 13th minute.
Lloyd James sent in a deep cross from the right wing, which found its way to the far post.
Wright-Phillips jumped the highest and was able to send a downward header back across goal into the far corner of the net.
Four minutes after the break he added a second after taking on and beating Reading full-back Liam Rosenior. He created just enough space to send in a fierce drive, which beat Marcus Hahnemann.
Reading boss Steve Coppell brought Czech international Marek Matejovsky into midfield in place of Kalifa Cisse in the second-half and Reading looked better for it.
In the 57th minute Matejovsky set Noel Hunt free down the left wing and he set up a chance for Kevin Doyle. The Championship's leading scorer saw his shot parried by Kelvin Davis and the ball rebounded to Jimmy Kebe, who scored his first goal of the season.
A minute from time Reading came close to grabbing an equaliser but Doyle headed over from close range.
Reading were fortunate to escape two penalty appeals in the first half, both rejected by referee Lee Probert. The first came in the 19th minute when the ball struck Ivar Ingimarsson's arm, but any claims were waved away.
In the 23rd minute Hahnemann and Jordan Robertson went for the ball together. Reading's keeper appeared to touch it a split second before the Saints striker, who went crashing to the ground. Again the referee was unimpressed.
Poortvliet said: "I am so very proud of what we saw today. Reading are a good team, but we deserved to win.
"In the first half particularly we created a lot of chances and this win will create a lot of confidence.
"Last week we played Wolves and I thought we deserved more than we got. It's good that we can now look forward to the next game.
"In the past we have thought Bradley Wright-Phillips was better coming on as a substitute than starting so we told him 'come on Bradley' and he responded.
"Everybody can see what we are capable of. We work on it every day and every week and I think we will be a stronger team next year if we can keep our young players."
Reading manager Coppell said: "We never do well after an international break, but I am at a loss to work out why we started so slowly.
"I don't believe we underestimated Southampton because all week I have been stressing that we are not good enough to underestimate anybody.
"They could have scored more, especially in the first half and there were a lot of poor performances from us today.
"They played good football, scored two good goals and they could have had more."