Adipocyte differentiation is a key process in determining the number of mature adipocytes in the development of obesity. Here, we examined the function of curcumin, a dietary polyphenol found in turmeric, and its underlying mechanisms in adipocyte differentiation. Our study reveals that curcumin exerts an anti-adipogenic function both in 3T3-L1 murine cells and in human primary preadipocytes as determined by intracellular lipid accumulation assay, quantitative analysis of adipocyte marker gene expression and a noninvasive multimodal Coherent Anti-Stokes Raman Scattering (CARS) microscopic analysis of intracellular curcumin. The inhibitory action of curcumin was largely limited to the early stage of adipocyte differentiation, where curcumin was found to inhibit mitotic clonal expansion (MCE) process as evidenced by impaired proliferation, cell-cycle entry into S phase and the S to G2/M phase transition of confluent cells, and levels of cell cycle-regulating proteins with no significant effect on cell viability and cytotoxicity. This, in turn, resulted in inhibition of mRNA levels of early adipogenic transcription factors, particularly Krüppel-like factor 5 (KLF5), CCAAT/enhancer binding proteinα (C/EBPα) and peroxisome proliferator-activated receptorγ (PPARγ), in the early stage of adipocyte differentiation. Supplementation with rosiglitazone, a PPARγ ligand, during the early stage of adipocyte differentiation partially rescued curcumin-inhibited adipocyte differentiation. Collectively, our results show that curcumin is an anti-adipogenic dietary bioactive component largely involved in the modulation of the MCE process during the early stage of adipocyte differentiation.