FoxO1 and C/EBPβ are important transcription factors during adipogenic differentiation; however, the potential mechanisms of their inter-regulation in adipogenesis remain unknown. We found that FoxO1 and C/EBPβ are abundant in adipose tissues of 3- and 180-day pigs at the mRNA and protein levels. During porcine preadipocyte differentiation, C/EBPβ expression increases to the peak at Day 2 and then decreases. In contrast, FoxO1 is lowest on Day 2 and gradually increases. Knockdown of FoxO1 or C/EBPβ with lentivirus-mediated shRNA enhances or inhibits lipid accumulation and adipogenic maker (C/EBPα and aP2) expression, respectively. FoxO1 depletion causes a mild decrease of C/EBPβ protein level, and C/EBPβ interference also inhibits the expression of FoxO1 protein. Knockdown of both FoxO1 and C/EBPβ promotes lipid accumulation at Day 8, and increases the adipogenic markers during differentiation in comparison with the controls and the FoxO1 knockdown alone. Co-immunoprecipitation experiments indicate that FoxO1 binds to C/EBPβ in adipose tissues and in vitro. In conclusion, the results suggest that FoxO1 and C/EBPβ regulate preadipocyte adipogenesis possibly through C/EBPβ → FoxO1 → C/EBPβ feedback regulatory loop and FoxO1-C/EBPβ protein complex.