Conor Shaughnessy and Colby Bishop scored for Portsmouth late in the first half at Fratton Park to condemn Ipswich to their first loss in 10 matches.
Shaughnessy set Portsmouth on course to move four points clear of the relegation zone by heading home Adrian Segecic's corner in the 42nd minute.
Bishop doubled the lead just two minutes later, slotting a loose ball into the bottom corner from close range.
Second-placed Ipswich remain two points above third-placed Millwall after a defeat that erased the momentum from last weekend's derby win at Norwich.
At St Mary's, Southampton extended their unbeaten run to 18 games with a seventh win in a row, leaving them just three points behind Ipswich.
First-half goals from Cyle Larin and Ryan Manning and a late strike from Cameron Archer extended Southampton's blistering run.
Tonda Eckert's side are up to fourth place, with an FA Cup semi-final against Manchester City and a crucial home game with Ipswich on the agenda in a potentially magical end to the season.
Ipswich still have a game in hand on Southampton, but Saints supporters are starting to dream of returning to the Premier League after last season's relegation.