On 8 July 2016, Mário Rui was signed by A.S. Roma in a temporary deal for a loan fee of €3 million (plus €1.5 million in bonuses), with a conditional obligation to sign him outright for an additional €6 million. He started the season injured, only making his debut on 19 January 2017 when he played 78 minutes in a 4–0 home win over U.C. Sampdoria for the Coppa Italia.