Adiponectin, an anti-inflammatory and insulin-sensitizing protein secreted from adipose tissue, may be modulated by dietary fatty acids, although the mechanism is not fully known. Our objective was to investigate the effect of long-chain n-3 polyunsaturated fatty acids (PUFAs) on adiponectin in cultured human adipocytes, and to elucidate the role of peroxisome proliferator-activated receptor-γ (PPARγ) in this regulation. Isolated human adipocytes were cultured for 48 h with 100 µmol/l eicosapentaenoic acid (C20:5n-3, EPA), docosahexaenoic acid (C22:6n-3, DHA), palmitic acid (C16:0), 100 µmol/l EPA plus 100 µmol/l DHA, or bovine serum albumin (control). Additionally, adipocytes were treated for 48 h with a PPARγ antagonist (BADGE) or agonist (rosiglitazone) in isolation or in conjunction with either EPA or DHA. At 48 h, EPA and DHA increased (P < 0.05) adiponectin secretion by 88 and 47%, respectively, while EPA, but not DHA, also increased (136%, P < 0.001) cellular adiponectin protein. Interestingly, PPARγ antagonism completely abolished the DHA-mediated increase in secreted adiponectin, but only partially attenuated the EPA-mediated response. Thus, EPA's effects on adiponectin do not appear to be entirely PPARγ mediated. Rosiglitazone increased (P < 0.001) the secreted and cellular adiponectin protein (90 and 582%, respectively). Finally, the effects of EPA and rosiglitazone on adiponectin secretion were additive (+230% at 48 h combined, compared to 121 and 124% by EPA or rosiglitazone alone, respectively). Overall, our findings emphasize the therapeutic importance of long-chain n-3 PUFA alone, or in combination with a PPARγ agonist, as a stimulator of adiponectin, a key adipokine involved in obesity and related diseases.