Background: CTRP3, a novel adipokine, has been linked with a variety of physiological functions, including adipokines secretion, energy metabolism, through an endocrine mean. This study evaluated the role of serum CTRP3 levels in the development of nonalcoholic fatty liver disease (NAFLD). Methods: The hospital-based longitudinal study recruited urban residents who took health examination. Serum CTRP3 levels were evaluated by an enzyme-linked immunosorbent assay. The adjusted odds ratio (OR) and 95% confidence interval (CI) were calculated to assess the relationship between baseline serum CTRP3 and incidence of NAFLD at follow-up. Results: Of the 814 participants at baseline, 313 subjects were included at follow-up. At baseline, serum CTRP3 level was lower in subjects with NAFLD (283.3 [159.6-375.0] ng/ml) than it in non-NAFLD subjects (295.0 [184.0-398.0] ng/ml) (p = 0.006). Meanwhile, serum CTRP3 level was inversely correlated with body mass index, waist-to-hip ratio, triglycerides and fasting plasma glucose. After a 3-y follow-up, the CTRP3 concentrations decreased from the baseline (206.7 [136.3-322.6] ng/ml) to the follow-up (177.4 [112.1-295.5] ng/ml, p < 0.001) in the subjects who developed NAFLD (n = 55). Compared with the 1st Quartile of baseline serum CTRP3, the subjects in the 3rd Quartile and 4th Quartile indicated lower risks of NAFLD progression at 3-y (adjusted OR = 0.451, 95% CI [0.270-0.755], p = 0.002 and adjusted OR = 0.468, 95% CI [0.310-0.707], p < 0.001). Conclusion: Serum level of CTRP3 was inversely associated with the progress of NAFLD independently at 3-y.