Chinese culture attaches great importance to the education and cultivation of youth conscientiousness, however in the context of Chinese culture, little is known about the relationship between conscientiousness and mental and physical health. The present study aimed to investigate whether there is a reciprocal relationship between conscientiousness and well-being (subjective and physical well-being) among Chinese undergraduate students. A series of self-reported questionnaires were administered to 365 undergraduate students in 2 waves, separated by 1 year. Cross-lagged regression analyses were applied to examine the reciprocal relationships. Results indicated that conscientiousness positively predicted subsequent levels of positive affect and life satisfaction, while negatively predicted subsequent levels of negative affect and physical symptoms, controlling for the effects of gender, age, body-mass index, socioeconomic status, and the prior level of conscientiousness. Whereas, positive and negative affect, life satisfaction, and physical symptoms did not significantly predict subsequent levels of conscientiousness. This study suggests that conscientiousness is a robust and prospective predictor of subjective and physical well-being. The reciprocal relationship between conscientiousness and well-being was not confirmed in the current sample of Chinese undergraduate students.