Mice lacking phosphatidylethanolamine N-methyltransferase (PEMT, Pemt^−/− mice) are resistant to high-fat diet (HFD)-induced obesity (DIO) but develop non-alcoholic steatohepatitis. PEMT expression is strongly induced during differentiation of 3T3-L1 adipocytes. Hence, we hypothesized that white adipose tissue (WAT) might be a key player in the protection against DIO in Pemt^−/− mice. We fed Pemt^−/− and Pemt^+/+ mice the HFD for 2 weeks, after which we examined adipocyte differentiation, adipogenesis and lipolysis in WAT. Pemt^−/− mice gained less body weight, had reduced WAT mass and had smaller adipocytes than Pemt^+/+ mice. The protein levels of adipose differentiation markers FABP4, PPARγ and C/EBPβ were not altered by genotype, but acetyl-CoA carboxylase expression and activation was reduced in the Pemt^−/− mice. The in vivo conversion of [¹⁴C]acetate to [¹⁴C]TG in WAT was also lower in Pemt^−/− mice. The release of glycerol from WAT explants was comparable between Pemt^+/+ and Pemt^−/− mice under basal condition and in the presence of isoproterenol, indicating unaffected lipolytic capacity. Furthermore, the amounts of leptin, cytokines and chemokines in WAT were not altered by genotype in mice fed the HFD for 2 weeks. However, after 10 weeks of HFD, WAT from Pemt^−/− mice had dramatically lower leptin, inflammatory cytokines (IL-1 and TNF-α) and chemokines (MCP-1 and RANTES), and significantly higher anti-inflammatory cytokine IL-10 than Pemt^+/+ mice. Together, our data show that PEMT deficiency did not affect the capability for differentiation and lipolysis in WAT. Decreased lipogenesis in WAT may contribute to the resistance to DIO in Pemt^−/− mice.