Objectives Accumulated evidence implies that night shift work may trigger liver dysfunction. Non-alcoholic fatty liver (NAFL) is suggested to be a necessary mediator in this process. This study aimed to examine the relationship between night shift work and elevated level of alanine transaminase (e-ALT) of workers and investigate the potential mediation effect of NAFL. Methods This study included all male workers from the baseline survey of a cohort of night shift workers. Information on demographics, lifestyle and lifetime working schedule was collected by face-to-face interview. Liver sonography was used to identify NAFL cases. Serum ALT level was detected by an automatic biochemical analyser. e-ALT was defined as ALT >40 U/L. Logistic regression models were used to evaluate ORs, and mediation analysis was employed to examine the mediation effect. Results Among 4740 male workers, 39.5% were night shift workers. Night shift workers had an increased risk of e-ALT (OR, 1.19, 95% CI 1.00 to 1.42). With the increase in night shift years, the OR of e-ALT increased from 1.03 (95% CI 0.77 to 1.36) to 1.60 (95% CI 1.08 to 2.39) among workers without NAFL. A similar trend was not found among workers with NAFL. In addition, no significant mediation effect of NAFL in the association between night shift work and e-ALT was found. Conclusions Night shift work is positively associated with abnormal liver function, in particular among workers without NAFL. Shift work involving circadian disruption is likely to exert a direct effect on liver dysfunction rather than rely on the mediation effect of NAFL.