Kochujang (Korean fermented red pepper paste) is a mixture of fermented soybeans, wheat, and red pepper powder. Kochujang has been reported to reduce body fat gain and lipid levels of adipose tissues and serum in rats. We studied the inhibitory effect of Kochujang on lipid accumulation and investigated the molecular mechanism of the action in 3T3-L1 adipocytes by measuring the expression levels of adipocyte-specific genes by real-time reverse transcription-polymerase chain reaction. When 3T3-L1 adipocytes were treated with Kochujang extract (KE), the sizes of adipocytes and leptin secretion were decreased. Hormone-sensitive lipase (HSL) was transcriptionally up-regulated at 4 hours, and glycerol secretion was increased at both 4 hours and 24 hours. Moreover, mRNA expression levels of both sterol regulatory element-binding protein 1-c (SREBP-1c) and peroxisome proliferator-activated receptor-γ (PPAR-γ), which are critical transcription factors for adipogenesis, were markedly down-regulated. Tumor necrosis factor-α (TNF-α) is reported to impair pre-adipocyte differentiation and induce lipolysis and apoptosis. KE treatment of 3T3-L1 adipocytes decreased TNF-α mRNA levels, but had no apparent affect on apoptosis. Taken together, our study shows that Kochujang decreased lipid accumulation in 3T3-L1 adipocytes by inhibiting adipogenesis through down-regulation of SREBP-1c and PPAR-γ and by stimulation of lipolysis due to increased HSL activity. TNF-α might not be involved in the reduction of lipid accumulation by KE.