Paeonol can regulate a variety of physiological and pathological processes such as thrombosis, oxidative stress, inflammation and atherosclerosis. However, its potential role and underlying mechanisms in obesity and lipid metabolism remain to be elucidated. In the present study, 3T3-L1 cells were differentiated and collected on days 4, 6 and 8. The expression levels of fatty-acid-binding protein 4 (FABP4) and microRNA (miR)-21 were detected using reverse transcription-quantitative PCR and western blot analyses. Cell viability was assessed using a Cell Counting Kit-8 assay. A miR-21 mimic was constructed and transfected into 3T3-L1 preadipocytes. Adipocyte differentiation was detected using Oil Red O staining. The proteins CD36, glucose transporter 4, peroxisome proliferator-activated receptor gamma (PPAR-gamma) and adipocyte protein 2 (Ap2) were detected using western blot analysis. The expression levels of FABP4 and miR-21 were increased in differentiated 3T3-L1 cells. Paeonol exhibited no effects on cell activity, whereas it inhibited the expression levels of miR-21 in the 3T3-L1 differentiated adipocytes. Paeonol suppressed the differentiation of 3T3-L1 adipocytes and its effect was partially reversed by the overexpression of miR-21. In addition, paeonol promoted the lipid degradation of 3T3-L1 adipocytes, increased the expression levels of PPAR-gamma and Ap2, and suppressed triglyceride synthesis in these cells. These effects were partially reversed by the overexpression of miR-21. In conclusion, the findings of the present study indicated that paeonol may exert protective effects against lipid formation and promote lipid degradation in adipocytes. These data provide evidence of the regulatory effect of paeonol on adipocyte differentiation and highlight its pathological significance.