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Objective To explore the mediating role of medical staff's autonomous motivation in the association between job requirements and job performance and to provide countermeasures and suggestions for improving medical staff's job performance. Methods By the convenience sampling method, 901 medical staff from 6 hospitals in Hangzhou were surveyed with the job demand scale, the autonomous motivation scale, and the job performance scale. The structural equation model was used to analyze the direct and indirect effects of job demand and autonomous motivation on job performance. Results Finally, 813 valid questionnaires were collected, with an effective recovery rate of 90.2%. In terms of job performance, the married medical staff scored higher, while those without a professional title or with working time less than 5 years scored lower (P < 0.05). The job requirements had a direct impact on job performance (β = 0.30, P < 0.01), which was indirectly affected through autonomous motivation (β = 0.069, P < 0.01), and the indirect effect percentage was 18.70%. Conclusions Autonomous motivation had a positive mediating effect on the association between job demands and job performance. Hospital managers should pay more attention to the working feelings of medical staff and give targeted guidance to medical staff with different levels of motivation to improve their autonomous motivation and, furthermore, to improve their work performance. [ABSTRACT FROM AUTHOR]