The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti-adipogenic action of n-6 PUFAs was dependent on a cAMP-dependent protein kinase-mediated induction of cyclooxygenase expression and activity. We show that n-6 PUFAs were pro-adipogenic when combined with a high carbohydrate diet, but non-adipogenic when combined with a high protein diet in mice. The high protein diet increased the glucagon/insulin ratio, leading to elevated cAMP-dependent signaling and induction of cyclooxygenase-mediated prostaglandin synthesis. Mice fed the high protein diet had a markedly lower feed efficiency than mice fed the high carbohydrate diet. Yet, oxygen consumption and apparent heat production were similar. Mice on a high protein diet had increased hepatic expression of PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator 1alpha) and genes involved in energy-demanding processes like urea synthesis and gluconeogenesis. We conclude that cAMP signaling is pivotal in regulating the adipogenic effect of n-6 PUFAs and that diet-induced differences in cAMP levels may explain the ability of n-6 PUFAs to either enhance or counteract adipogenesis and obesity.