Born in Sines, Setúbal District, Mário Rui started his career at local Vasco da Gama Atlético Clube.

He then played for the youth teams of Sporting CP, Valencia and Benfica,[5] being signed by Parma in summer 2011[6] and being loaned immediately to fellow Italians Gubbio;[7][8] he joined the Serie B club following that year's FIFA U-20 World Cup,[9] with the international transfer certificate arriving on 2 September.

[18] He started the season injured,[19] only making his debut on 19 January 2017 when he played 78 minutes in a 4–0 home win over Sampdoria in the Coppa Italia.

[24] He became the starting left-back after Faouzi Ghoulam's knee injury in November,[25] and in April his agent threatened to find him a new team if he would not retain that place after the Algeria international's recovery.

[26] Mário Rui played the first 81 minutes of the 2020 Coppa Italia final on 17 June, which Napoli won on penalties after a goalless draw with Juventus.

During the 2011 FIFA U-20 World Cup he appeared in five out of seven matches for the eventual finalists,[33][34] scoring his only goal for his country in a 1–0 group stage victory over New Zealand.