Backgrounds: Night-shift workers are exposed to nocturnal light and are more prone to circadian rhythm disorders. Although night-shift work is thought to be associated with the decrease in melatonin secre-tion, studies have shown inconsistent results. Methods: This systematic review and meta-analysis studied the association between night-shift work and melatonin levels. Pubmed and Embase databases were used for literature searching. The pooled standardized mean differences (SMDs) and 95% confidence intervals (CIs) were used to compare the differences between night-shift workers and the controls. Results: Thirty-three studies reported in 25 articles (1845 night-shift workers and 3414 controls, mean age 45.12 years) were included after a systematic literature review. Data of circulating melatonin levels and its metabolites, 6-sulfatoxymelatonin (aMT6s) in urine were collected for meta-analysis. The results showed that the first morning-void aMT6s level in night-shift workers was significantly lower than in day workers (SMD = -0.101, 95% CI = -0.179 to-0.022, P = 0.012). The level of mean 24-h urinary aMT6s was lower in night-shift workers than day workers (SMD: -0.264, 95% CI: -0.473 to -0.056, P = 0.013). Among fixed night-shift workers, the level of circulating melatonin, as well as first morning void aMT6s was lower than that of day workers. Conclusion: Our findings indicate that experience of night-shift work is associated with suppression of melatonin production, especially among fixed night-shift workers. (C) 2020 Elsevier B.V. All rights reserved.