Dietary l-arginine supplementation decreases backfat thickness and increases intramuscular fat content in growing-finishing pigs, but the underlying mechanisms are unknown. In this study, the effect of arginine on differentiation of porcine intramuscular preadipocytes was investigated in vitro. We showed that the mRNA and protein expressions of the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α were upregulated by arginine supplementation. Furthermore, the intracellular triglyceride level was increased by arginine supplementation. We also showed that activation of the Wnt/β-catenin signal pathway by using lithium chloride (LiCl) significantly attenuated arginine-induced upregulation of PPARγ and increased the phospho-β-catenin level. These findings suggested that arginine promotes porcine intramuscular preadipocyte differentiation, which might be via repressing the Wnt/β-catenin signaling pathway.