Preadipocyte factor 1 (Pref-1), also known as a delta-like 1 protein, is a transmembrane and secreted protein containing the epidermal growth factor-like repeat. Pref-1 inhibits adipocyte differentiation by activating the ERK1/2 pathway. MicroRNAs, a new class of small noncoding RNAs of 20-24 nucleotides, act as negative regulators of gene expression and result in mRNA degradation or translational repression. MicroRNA-143 (miR-143) is known to induce adipocyte differentiation; however, miR-143 targets in the regulation of adipocyte differentiation remain unknown. In this study, we investigated whether pref-1 is a miR-143 target to regulate adipogenesis. After the induction of adipocyte differentiation the level of miR-143 was increased, whereas the expression of pref-1 mRNA was decreased. The pref-1 protein level was also down-regulated in preadipocytes ectopically expressing miR-143, and recovered by miR-143 inhibitor. The binding region for miR-143 was predicted to be located between positions 247 and 252 in the 3'-UTR of pref-1. The luciferase activity of the vector containing the wild-type 3'-UTR of pref-1 was decreased by 65 % in cells transfected with miR-143 mimic compared to that of the corresponding control. In contrast, the activity of the pref-1 mutant cells was not affected by the treatment with miR-143 mimic. The ectopic expression of miR-143 mimic suppressed the phosphorylation of ERK1/2 induced by pref-1 in 3T3-L1 cells. However, the suppressed phosphorylation was restored by miR-143 inhibitor. Taken together, these data suggest that miR-143 promotes adipogenesis by directly modulating the pref-1 expression in adipocytes.