Obesity is characterized by the excessive accumulation of fat to adipose tissue, which is related to abnormal increasing white adipose tissue (WAT) in the body, and it upregulates the risk of multiple diseases. Here, kuanoniamine C, which is a pyridoacridine alkaloid, suppressed the differentiation of pre-adipose cells into white adipocytes via the modulation of mitochondrial function, and inhibited WAT expansion in the early phase of high-fat-diet-induced obesity model. Pharmacological analysis revealed that inhibition of mitochondrial respiratory complex II, which new target of kuanoniamine C, activated reactive oxygen species (ROS)-extracellular signal-regulated kinase (ERK)-beta-catenin signaling, and this signaling was antagonized by insulin-, IBMX-, and dexamethasone-induced adipogenesis. Therefore, the kuanoniamine C might prevent abnormal WAT expansion even when eating a diet that is not calorie restricted.