MicroRNAs (miRNAs), as a series of important short-chain non-coding RNAs, play an important post-transcriptional role in many biological activities, including adipogenesis. miR-144 is significantly upregulated in type II diabetes (T2D), and is considered to be an important biomarker for T2D. However, although the occurrence of T2D is inextricably linked to adipogenesis, whether miR-144 directly regulates adipogenesis remains to be further explored. In this paper, we demonstrate that miR-144 has a higher expression level in a porcine high backfat group, and it has a significant positive effect on promoting the differentiation of pre-adipocytes. FoxO1 is a target gene of miR-144, and inhibits the differentiation of pre-adipocytes. On the other hand, we demonstrate that FoxO1 can bind to the AdipoQ gene promoter, then regulate the AdipoQ expression by binding to the FoxO1 binding site in the AdipoQ promoter -1,499 to -1,489 bp and -1,238 to -1,228 bp regions, especially the -1,499 to -1,489 bp region. Meanwhile, miR-144 and FoxO1 co-expressional research has also shown that both factors regulate adipogenesis. To sum up, our research indicates that miR-144 targets FoxO1, thus reducing its expression and inhibiting its promotional effect on adiponectin, thereby alleviating the inhibitory effect of adiponectin on adipogenesis.