Excessive adipogenesis in adipocytes results in obesity. Berberine, a natural isoquinoline alkaloid, has antiobesity properties. However, the underlying molecular mechanisms have remained unclear up to now. In this study, porcine adipocytes were cultured and treated with berberine. Cellular lipid content was measured by Oil Red O staining extraction. The role of an adenosine monophosphate-activated protein kinase (AMPK) signaling pathway was evaluated by the phosphorylation detection of AMPKα protein and knockdown of AMPK alpha1 (Ampka1) gene. Gene expressions were analyzed by Western blot and real-time reverse transcription-polymerase chain reaction (RT-PCR). The results showed that berberine reduced lipid accumulation in porcine adipocytes in a dose- and time-dependent manner and increased phosphorylation of AMPKα. Furthermore, berberine significantly downregulated the mRNA expression of related genes to adipogenesis including peroxisome proliferator activated receptor gamma 2 (Pparg2), CCAAT/enhancer-binding protein alpha (Cebpa), Cebp beta (Cebpb), sterol regulatory element binding transcription factor 1 (Srebf1), acetyl-CoA carboxylase-1 (Acc-1), fatty acid synthase (Fas), fatty acid binding protein 4 (Fabp4), and stearoyl-CoA desaturase 1 (Scd1). Knockdown of Ampka1 markedly reversed the inhibitory effect of berberine on lipid accumulation and mRNA expression of the above genes except Cebpb in porcine adipocytes. Meanwhile, the protein expression of these adipogenic genes in response to berberine and Ampka1 knockdown paralleled the alterations of their mRNA level. These results suggest that berberine inhibits adipogenesis in porcine adipocytes via AMPK-dependent and -independent multiple mechanisms, which would provide an important idea for the reduction of porcine body fat, as well as the prevention and treatment of human obesity.