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Passiflora incarnataL. is known to contain abundant C-glycosyl flavones, which exhibit various biological activities; however, its anti-obesity effects have not yet been investigated. To this end, herein, we conducted UPLC-MS/MS to identify flavonoids in Passiflora incarnataL. aqueous and 60% ethanol extracts (denoted as PAE and PEE, respectively) and HPLC to quantify five major C-glycosyl flavones (vitexin, isovitexin, orientin, isoorientin, and vicenin-2). PEE contained most of the C-glycosyl flavones compared to PAE. The analysis of the inhibitory effect on lipid accumulation of these extracts and the five C-glycosyl flavones were conducted using differentiated 3T3-L1 and oleic acid (OA)-treated HepG2 cells. PEE more effectively inhibited lipid accumulation than PAE. Among C-glycosyl flavones, vicenin-2 showed the strongest lipid accumulation inhibitory effect in both cells and significantly inhibited lipid-metabolism-related FAS, PPARγ, FABP4, and SREBP1c. Moreover, we fed a high-fat diet (HFD) containing PEE to C57BL/6 mice for eight weeks and measured the body weight, serum lipid, hepatic lipid, and adipocyte size. The results showed that PEE reduced body weight gain and serum lipids in HFD-fed mice. PEE suppressed hepatic lipid accumulation and adipocyte size by suppressing adipogenesis and lipogenesis. Our results demonstrated that C-glycosyl-flavone-rich PEE can be used as an anti-obesity agent.