Based on our previous solexa sequencing data, combining with bioinformatics analysis of microRNA expression changes in porcine adipose tissue from different growth phases in a model of obesity, we found the expression level of miR-191 was significantly different in developing swine adipose tissue. In this research, we successfully overexpressed miR-191's transcripts by recombinant adenovirus in primary cultured porcine preadipocytes, and then investigated the impact on differentiation of pig preadipocyte by Real-time quantitative PCR, Western blot. The results showed that miR-191's expression level gradually increased during differentiation. The miR-191 transcripts increased dramatically when miR-191 overexpressed compared to the control group, which caused the decrease of C/EBPβ, PPARγ and aP2's mRNA level and the sequentially repression of preadipocytes differentiation. Meanwhile, Western blotting results indicated that the expression of C/EBPβ protein in primary cultured porcine preadipocytes that overexpressed miR-191 was 55% lower than that in control cells after 48 h post-transfection. Importantly, we predicted that C/EBPβ was miR-191's target gene through bioinformatics software TargetScan and MicroInspector. This is conformed by dual luciferase reporter vector system assay which showed that miR-191 can target C/EBPβ 3′ untranslated region (3′UTR) directly and then decrease its expression. In conclusion, this study indicates that miR-191 can attenuate pig preadipocytes defferetiation possibly by repressing the gene expression of C/EBPβ which is one of the early marker gene of adipocytes differentiation.