Adiponectin is an important adipose-specific protein, which possesses insulin (INS)-sensitizing, antiinflammatory, and antiatherosclerotic functions. However, its regulation remains largely unknown. In this study, we identified that ryanodine receptor (RyR)3 plays an important role in the regulation of adiponectin expression. RyR3 was expressed in 3T3-L1 preadipocytes, and its level was decreased upon adipogenesis. Silencing of RyR3 expression in 3T3-L1 preadipocytes resulted in up-regulated adiponectin promoter activity, enhanced adiponectin mRNA expression, and more adiponectin protein secreted into the medium. An inverse relation between RyR3 and adiponectin mRNA levels was also observed in adipose tissues of db/db mice. In addition, knockdown of RyR3 with small interfering RNA (siRNA) in db/db mice and high-fat diet-fed obese mice increased serum adiponectin level, improved INS sensitivity, and lowered fasting glucose levels. These effects were in parallel with decreased mitochondrial Ca(2+), increased mitochondrial mass, and reduced activating transcription factor 3 (atf3) expression. Overexpression of atf3 in 3T3-L1 preadipocytes blocked the effect of RyR3 silencing on adiponectin expression, indicating that an atf3-dependent pathway mediates the effect downstream of RyR3 silencing. Our data suggest that RyR3 may be a new therapeutic target for improving INS sensitivity and related metabolic disorders.