OBJECTIVES: Previous research has indicated that shift workers exhibit fewer healthy behaviors than those working regular daytime hours. Although health literacy influences health behaviors, studies comparing health literacy levels between shift and fixed-day workers and investigating whether differences in health behaviors between these 2 groups are mediated by health literacy are lacking. METHODS: This cross-sectional study used a self-administered questionnaire in a large-scale manufacturing company. Overall, 2152 manual workers aged 18-64 years were enrolled in this study (961 and 1191 fixed-day and shift workers, respectively). Logistic regression structural equation models were used for analyzing the mediating role of health literacy in the relationship between shift work and health behaviors. RESULTS: Shift and fixed-day workers did not show differences in age-adjusted health literacy. Compared with fixed-day workers, the odds ratios of shift workers for leisure time exercise, not currently smoking, having breakfast, brushing their teeth, and eating green and yellow vegetables were 0.85 (95% CI, 0.70-1.02), 0.68 (95% CI, 0.56-0.80), 0.63 (95% CI, 0.52-0.76), 0.79 (95% CI, 0.65-0.95), and 0.58 (95% CI, 0.48-0.70), respectively. Mediation analysis showed that the odds ratios of the direct effects of shift work on favorable habits were almost the same as the total effect. CONCLUSIONS: This study observed that health literacy did not mediate health behavior and shift work. Further research is needed to clarify the causes of these differences.