Objective Circulating miR-29b has been rEPORted to be positively correlated with nonalcoholic fatty liver disease (NAFLD). However, the role of miR-29b-3pI in NAFLD progression is unclear. The purpose of this study was to evaluate the expression of miR-29b-3p in NAFLD models and to identify the potential functions of miR-29b-3p in lipid accumulation and fibrosis in hepatocytes. Methods Palmitic acid (PA)-treated L02 cells were used as an in vitro cellular model of NAFLD. miR-29b-3p and insulin-like growth factor-1 (IGF-1) expression levels were determined by RT-qPCR or Western blot. miR-29b-3p mimic / inhibitor or IGF-1 siRNA were transfected into L02 cells exposed to PA. Lipid accumulation was determined by oil red O staining, and triglyceride and total cholesterol assays. Direct interaction between miR-29b-3p and IGF-1 was determined by dual-luciferase reporter assay. Results The result revealed that miR-29b-3p was upregulated in our in vitro cellular model of NAFLD while IGF-1 concentrations decreased. miR-29b-3p inhibition significantly suppressed lipid accumulation and fibrosis in PA-treated L02 cells. miR-29b-3p targets IGF-1 and suppresses its expression in vitro. Interestingly, the effects of miR-29b-3p overexpression on lipid accumulation and fibrosis in PA- treated L02 cells was enhanced by IGF-1 silencing. Conclusions Our result suggested that miR-29b-3p has a negative regulatory effect on lipid accumulation and fibrosis in hepatocytes by targeting IGF-1. This study provides evidence that miR-29b-3p might be a promising therapeutic target for NAFLD.