Objective To explore the mediating effect of fatigue on the relationship between negative emotion and burnout in military medical university students. Methods By random sampling, depression anxiety stress scale 21 (DASS- 21), multidimensional fatigue questionnaire (MFI-20) and Maslach burnout questionnaire (MBI) were used to investigate the negative emotion, fatigue and burnout of five-year undergraduates of clinical medicine in a military medical university. Pearson product moment correlation and multivariate hierarchical regression were used to analyze the relationships among different variables. Structural equation model was constructed and Bootstrap test was used to analyze the mediating effect.Results A total of 270 questionnaires were distributed, and 250 valid questionnaires were enrolled, with a response rate of 92.6%. The scores of three dimensions (depression, anxiety and stress) of negative emotion were significantly higher than the norm (all P<0.01). The score of physical fatigue was significantly higher than the norm (P<0.01), and the scores of mental fatigue, power loss and activity reduction were significantly lower than the norm (P<0.05, P<0.01). There were no differences between the scores of emotional exhaustion and cynicism and the norm, but the low achievement score was significantly higher than the norm (P<0.01). There were significant correlations between negative emotion, fatigue and burnout in all dimensions (all P<0.01). Multivariate hierarchical regression analysis showed that fatigue could explain 53.3% of variance variation of burnout (F=71.140, P<0.01). Both physical fatigue and mental fatigue had positive predictive effects on burnout (β=0.516, P<0.01; β=0.188, P<0.01). On the basis of fatigue, negative emotion could independently explain 1.9% of variance variation of burnout (F=44.243, P<0.01). Anxiety, depression and stress could explain 33.5% of the variation variance of burnout (F=42.333, P<0.01), and depression and stress had positive predictive effects on burnout (β=0.236, P<0.01; β=0.258, P<0.05). On the basis of negative emotion, fatigue could independently explain 21.7% of variance variation of burnout (F=44.243, P<0.01). Physical fatigue and mental fatigue had positive predictive effects on burnout (β=0.423, P<0.01; β=0.166, P<0.01). Mediation effect models and Bootstrap test results showed that negative emotion could indirectly affect burnout through fatigue (χ²=74.508, df=17, χ²/df=4.383, comparative fit index[CFI]=0.955, incremental fit index[IFI]=0.956, relative fit index[RFI]=0.943, Tucker-Lewis index[TLI]=0.926). The mediating effect of fatigue was 65.3%. Conclusion Fatigue plays a mediating role between negative emotion and burnout. The military medical students with high anxiety, depression and stress need more attention and effective measures should be adopted to avoid the aggravation of their fatigue and burnout.