Aims To investigate the intracellular lipid accumulation inhibitory effect of spent culture medium extract and the cytoplasmic fraction of Weissella koreensis OK1‐6 cells isolated from kimchi in differentiating 3T3‐L1 cells. Methods and Results Differentiating 3T3‐L1 cells were treated with either cytoplasmic fraction of W. koreensis OK1‐6 cells or its spent media for 4 days. Both the spent culture medium extract and cytoplasmic fraction of W. koreensis OK1‐6 cells significantly decreased the triglyceride concentration and intracellular lipid accumulation in the treated groups compared with the control group. The mRNA expression levels of C/EBP‐α, one of the major transcriptional factors involved in adipocyte differentiation, were significantly less expressed in 3T3‐L1 cells treated with the spent medium and cytoplasmic fraction. The expressions of aP2, fatty acid synthase (FAS) and SREBP1 genes were also decreased significantly. Conclusions These results suggested that W. koreensis OK1‐6 could play a crucial role in preventing intracellular lipid accumulation by down‐regulating the expression of adipocyte‐specific genes C/EBPα, aP2, SREBP1 and FAS. Significance and Impact of the Study These results may contribute to nutraceutical and food industries in developing probiotic‐based therapies for the treatment and prevention of obesity.