The current study is based on the self-determination theory, which proposes that basic psychological needs fulfillment may account for the relationship between mindfulness and athletes’ well-being. Using a two-wave longitudinal design, in the current study, we recruited 104 college athletes (mean age 20.66 years) and facilitated mindfulness, basic psychological needs fulfillment, positive and negative affects, life satisfaction, and subjective vitality measurements. Multiple regressions during the interaction term revealed that Time 1 basic psychological needs fulfillment mediated the relationship between Time 1 mindfulness and Time 2 hedonic well-being (satisfaction with life, positive and negative affects) and Time 2 eudaemonic well-being (subjective vitality) in athletes. In addition, the mediational effect remained evenly controlled in the Time 1 well-being index. Our hypothesis was supported, and the results are discussed in terms of mindfulness and its application in sports.