Although accumulating evidence has revealed the effect of mindfulness training on the reduction of general stress and the improvement of mental well-being, the underlying mechanism remains unclear. In this study, we investigated whether interoceptive attention to respiratory signals plays a role. Healthy adults were randomly assigned to either receive an 8-week mindfulness training (n = 29) or inactive control (n = 28). The pre- and post-training self-reported states of negative mood were assessed together with an objective measure of interoceptive attention to respiration. Compared to the control group, mindfulness training led to a decrease in the level of negative mood and an increase in interoceptive sensitivity. Mediation analysis further showed that the effect of mindfulness training on the reduction of negative mood was fully mediated by increased interoceptive sensitivity. These results suggest that mindfulness training effectively alleviates negative mood by enhancing interoceptive attention to respiratory signals.