Claudio Ranieri's side looked to be returning to the summit after Francesco Totti's 14th-minute goal but a second-half double from Giampaolo Pazzini sent Samp back up to the fourth and final Champions League qualifying spot.
Inter, who had let slip a nine-point lead from February, beat Atalanta 3-1 on Saturday and lie two points ahead of Roma with three games left as they seek a fifth straight scudetto.
The visit of Samp had looked the toughest-remaining hurdle in debt-laden Roma's fairytale route to a surprise fourth Serie A title and so it proved.
Totti, substituted in last weekend's derby win over Lazio because he was over-excited, expertly turned in Mirko Vucinic's pass to boost calls for the Italian to come out of international retirement at June's World Cup.
First Pazzini headed in former Roma forward Antonio Cassano's cross early in the second half before poking in the winner five minutes from time.
"First half that wasn't us," Pazzini told Sky TV. "Then we sorted it out in the second half. It was a great match and our Champions League dream continues."
Iaquinta, likely to be in Italy's World Cup squad and possibly the starting lineup, has struggled with injury for most of the season but looked fresh after coming on as a substitute.
Bottom side Livorno were relegated despite a 3-1 home victory over Catania and Bologna's 2-1 win over Parma also meant Siena are all but down following a 4-1 defeat at Udinese.
