Objective: Human adenovirus Ad-36 causes adiposity in animal models and enhances differentiation and lipid accumulation in human and 3T3-L1 preadipocytes, which may, in part, explain the adipogenic effect of Ad-36. We determined the consequences of Ad-36 infection on leptin and glucose metabolism in fat cells. Design: 3T3-L1 preadipocytes were used to determine the effect of infection by human adenoviruses Ad-36, Ad-2, Ad-9 and Ad-37 on leptin secretion and lipid accumulation. Rat primary adipocytes were used to determine the effect of Ad-36 infection on leptin secretion and glucose uptake in vitro. Furthermore, the effect of Ad-36 on expressions of leptin and selected genes of de novo lipogenesis pathway of visceral adipose tissue were compared ex vivo, between Ad-36 infected and uninfected control rats. Results: Ad-36 suppressed the expression of leptin mRNA in 3T3-L1 cells by approximately 58 and 52% on days 3 and 5 post-infection, respectively. Leptin release normalized to cellular lipid content was 51% lower (P<0.002) in the Ad-36 infected 3T3-L1 cells. Lipid accumulation was significantly greater and leptin secretion was lower for the 3T3-L1 cells infected with other human adenoviruses Ad-9, Ad-36, or Ad-37. Whereas, human adenovirus Ad-2 did not influence cellular lipid accumulation or the leptin release. In rat primary adipocytes, Ad-36 reduced leptin release by about 40% in presence of 0.48 (P<0.01) or 1.6 nM insulin (P<0.05) and increased glucose uptake by 93% (P<0.001) or 18% (P<0.05) in presence of 0 or 0.48 nM insulin, respectively. Next, the adipose tissue of Ad-36 infected rats showed two to fivefold lower leptin mRNA expression, and 1.6- to 21-fold greater expressions for acetyl Co-A carboxylase-1 and 1.2- to 6.3-fold greater expressions for fatty acid synthase, key genes of de novo lipogenesis, compared to the uninfected weight and adiposity matched controls. Conclusion: The in vitro and ex vivo studies show that Ad-36 modulates adipocyte differentiation, leptin production and glucose metabolism. Whether such a modulation contributes to enhanced adipogenesis and consequent adiposity in Ad-36 infected animals or humans needs to be determined.