Background: This study examined relations between social support, job stress, and public service motivation, also assessed how social support and job stress affect public service motivation based on the job demands-resources (JD-R) theory. Methods: The survey investigated a sample of 973 healthcare workers employed in public hospitals in Beijing, Xiamen, and Guangzhou in 2017. Through random sampling according to employee number, age and job title, we selected 5% to 10% of healthcare workers in each target hospital. Finally, we got 973 valid participants, and the response rate was 93.8%. We used correlation analysis, structural equation modeling, Sobel test, and subgroup analysis to test by investigating a sample of 973 healthcare workers from 3 Chinese public hospitals. Results: Challenge stress and hindrance stress were directly inversely associated with public service motivation. Supervisor support was significantly positively associated with public service motivation, and the path from coworker support to public service motivation was significant. Supervisor support was significantly negatively associated with hindrance stress, and coworker support was significantly inversely associated with challenge stress. Hindrance stress and challenge stress significantly mediated the relations between supervisor support and public service motivation, and between coworker support and public service motivation respectively. Conclusions: Public service motivation might be raised by increasing supervisor support and coworker support and by limiting hindrance stress and challenge stress among healthcare workers in China.