Polymethoxyflavones are flavonoids that are abundant in citrus fruit peels and have beneficial effects on human health. Previous studies have demonstrated that the polymethoxyflavones, namely sudachitin and nobiletin, ameliorate obesity and diabetes in humans and rodents. Although nobiletin induces lipolysis in adipocytes, lipolytic pathway activation by sudachitin has not been clarified in adipocytes. In this study, the effect of sudachitin on lipolysis was elucidated in murine 3T3-L1 adipocytes. Glycerol release into the medium and activation of the cyclic AMP (cAMP)/protein kinase A (PKA)/hormone-sensitive lipase (HSL) pathway was evaluated in 3T3-L1-differentiated adipocytes. Treatment with sudachitin and nobiletin for 24 and 48 h did not induce cytotoxicity at concentrations of up to 50 μM. Sudachitin and nobiletin at concentrations of 30 and 50 μM increased intracellular cAMP and medium glycerol levels in 3T3-L1 adipocytes. Western blotting revealed that sudachitin and nobiletin dose-dependently increased protein levels of phosphorylated PKA substrates and phosphorylated HSL. Sudachitin- and nobiletin-induced glycerol release, phosphorylation of PKA substrates, and HSL phosphorylation were suppressed by pharmacological inhibition of adenylate cyclase and PKA. These findings indicated that sudachitin, similar to nobiletin, exerts anti-obesogenic effects, at least in part through the induction of lipolysis in adipocytes.