Valproic acid (VPA) has been used for the treatment of epilepsy and bipolar disorders for more than 30 yr. Obesity and insulin resistance are common side effects of VPA treatment. Adiponectin is an adipocyte-derived protein that plays an important role in controlling insulin sensitivity and glucose homeostasis. In this report, we examined the effects of VPA on adiponectin gene expression in C57BL/6J mice and in differentiated 3T3-L1 adipocytes. VPA treatment significantly decreased adiponectin protein and mRNA levels in both mice and 3T3-L1 adipocytes. The adipocyte study showed that VPA inhibited adiponectin gene expression in a dose- and time-dependent manner. Repression of adiponectin expression by VPA occurred at the transcription level and correlated with inhibition of histone deacetylase activity. Therapeutic concentrations of VPA increased overall histone acetylation and increased adiponectin promoter-driven luciferase expression in fibroblasts, but decreased adiponectin promoter activity in differentiated 3T3-L1 adipocytes. VPA treatment decreased adipogenic transcription factor CCAAT/enhancer binding protein-alpha (C/EBPalpha) levels and binding of C/EBPalpha to the adiponectin promoter without altering the levels of peroxisome proliferator-activated receptor-gamma and steroid regulatory element binding protein-1. Furthermore, VPA did not suppress adiponectin gene expression in C/EBPalpha gene-deficient adipocytes that stably expressed exogenous peroxisome proliferator-activated receptor-gamma2. Together, these results demonstrate that histone deacetylase inhibitor VPA suppresses adiponectin gene expression in mature adipocytes. The study also provides evidence that diminished C/EBPalpha protein level and decreased binding at the adiponectin promoter mediate the inhibitory effects of VPA on adiponectin gene transcription.