Objectives: This study explored the relationships between challenge stress, hindrance stress, and healthcare quality, and the mediating effect of public service motivation (PSM), among healthcare workers in Chinese public hospitals. Methods: Using widely adopted scales to measure all variables, we used descriptive statistical analysis, analysis of variance, correlation analysis, structural equation modeling and subgroup analysis to investigate data from 2066 healthcare workers in 68 Chinese public hospitals. Results: Challenge stress and hindrance stress were strongly correlated among the participants (β = 0.59; p < .001). Challenge stress was significantly and positively associated with PSM (β = 0.14; p < .001) and healthcare quality (β = 0.16; p < .001), and hindrance stress was significantly and negatively associated with PSM (β = -0.29; p < .001) and healthcare quality (β = -0.10; p < .001). PSM was directly positively associated with healthcare quality (β = 0.59; p < .001) and indirectly mediated the relationship of hindrance stress with healthcare quality. Conclusions: Healthcare quality might be improved by limiting hindrance stress, increasing PSM, and controlling challenge stress.