In current times, obesity is a major health problem closely associated with metabolic disease such as diabetes, dyslipidemia, and cardiovascular disease. The direct cause of obesity is known as an abnormal increase in fat cell size and the adipocyte pool. Hyperplasia, the increase in number of adipocytes, results from adipogenesis in which preadipocytes differentiate into mature adipocytes. Adipogenesis is regulated by local and systemic cues that alter transduction pathways and subsequent control of adipogenic transcription factors. Therefore, the regulation of adipogenesis is an important target for preventing obesity. Myonectin, a member of the CTRP family, is a type of myokine released by skeletal muscle cells. Although several studies have shown that myonectin is associated with lipid metabolism, the role of myonectin during adipogenesis is not known. Here, we demonstrate the role of myonectin during adipocyte differentiation of 3T3-L1 cells. We found that myonectin inhibits the adipogenesis of 3T3-L1 preadipocytes with a reduction in the expression of adipogenic transcription factors such as C/EBP alpha, beta and PPAR gamma. Furthermore, we show that myonectin has an inhibitory effect on adipogenesis through the regulation of the p38 MAPK pathway and CHOP. These findings suggest that myonectin may be a novel therapeutic target for the prevention of obesity.