Background: Presenteeism affects the performance of healthcare workers. This study examined associations between job stress, affective commitment, and presenteeism among healthcare workers. Methods: To investigate the relationship between job stress, affective commitment, and presenteeism, structural equation modeling was used to analyze a sample of 1392 healthcare workers from 11 Class A tertiary hospitals in eastern, central, and western China. The mediating effect of affective commitment on the association between job stress and presenteeism was examined with the Sobel test. Results: Job stress was high and the level of presenteeism was moderate among healthcare workers. Challenge stress and hindrance stress were strongly correlated (β = 0.62; p < 0.05). Affective commitment was significantly and directly inversely correlated with presenteeism (β = −0.27; p < 0.001). Challenge stress was significantly positively correlated with affective commitment (β = 0.15; p < 0.001) but not with presenteeism. Hindrance stress was significantly inversely correlated with affective commitment (β = −0.40; p < 0.001) but was significantly positively correlated with presenteeism (β = 0.26; p < 0.001). Conclusions: This study provides important empirical data on presenteeism among healthcare workers. Presenteeism can be addressed by increasing affective commitment and challenge stress and by limiting hindrance stress among healthcare workers in China.