Currently, the utilization of deep-sea water (DSW) is receiving much attention due to its high productivity, large quantity, and potential for biological application. The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days, confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. In this study, the effects of DSW on differentiation adipocyte 3T3-L1 cells were studied. DSW significantly decreased lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. DSW of hardness 1,000 was the most effective for inhibiting adipocyte differentiation without any cytotoxicity. DSW significantly reduced expression mRNA levels of PPARgamma and C/EBPalpha and protein levels of fatty-acid-binding protein and adiponectin. Our results suggest a potential role for DSW as anti-obesity agents by inhibiting adipocyte differentiation mediated through the down-regulated expression of adipogenic transcription factors and adipocyte-specific proteins.