Scores were level at 2-2 on aggregate after Atletico won the return leg 2-1 thanks to substitute Jhon Valoy's 93rd-minute goal that sent the match to penalties at the Estadio Atanasio Girardot.
And Junior were no match for Atletico Nacional in the shoot-out as Juan Carlos Osorio's men continued their stranglehold of Colombia's top-flight, having done the double last season.
Atletico Nacional went into the game needing a result following their 1-0 loss at Junior in the opening leg on Sunday.
The odds were stacked against them having lost four of their past five games in all competitions but they made the perfect start to proceedings thanks to veteran defender Alexis Henriquez, who opened the scoring in the second minute to level the tie on aggregate.
Atletico Nacional's joy was short-lived after Edison Toloza equalized for Junior in the 17th minute, which put the visitors' ahead 2-1 on aggregate.
And just as it seemed Junior, seven-time champions, would clinch their first title since 2011, Valoy pounced in the third minute of injury-time to send the match to penalties.
Atletico Nacional have now won 14 titles, the same amount as rivals Millonarios.
