We recently demonstrated that palmitoleic acid (C16:1n7), a monounsaturated fatty acid, increases the metabolic and oxidative capacity of 3T3-L1 adipocytes. Herein, the effect of 16:1n7 supplementation on metabolic parameters on white adipose tissue (WAT) and liver of obese mice induced by a high-fat diet (HFD) was addressed by analyzing metabolic (dys)function and altered genes expression in adipose tissue, as well as liver and serum biochemistry analysis. For this purpose, mice were induced to obesity for 8 weeks, and from the 5th week, they received 16:1n7 (300 mg/kg per day) or water for 30 days, by gavage. Subcutaneous inguinal (ING) and epididymal (EPI) WAT were removed for analysis of metabolic, (anti)inflammatory, adipogenic, and thermogenic genes expression by real-time reverse transcriptase–polymerase chain reaction. Additionally, metabolic activities of isolated adipocytes, such as glucose uptake, lipogenesis (triacylglycerol esterification), β-oxidation, and lipolysis in ING adipocytes, were also assessed. Despite the higher fat intake, the HFD group showed lower food intake but higher body weight, increased glucose, significant dyslipidemia, and increased liver and adipose depot mass, accompanied by liver steatosis. The 16:1n7 supplementation slowed down the body mass gain and prevented the increase of lipids in the liver. HFD+n7 animals presented increased fatty acid oxidation and lipogenesis compared to control, but no effect was observed on lipolysis and glucose uptake in ING isolated adipocytes. Besides, 16:1n7 increased the content of the mRNA encoding FABP4, but partially prevented the expression of genes encoding ATGL, HSL, perilipin, lipin, C/EBP-α, PPAR-γ, C/EBP-β, CPT1, NRF1, TFAM, PRDM16, and nitric oxide synthase 2 in ING depot from HFD group of animals. Finally, HFD increased Mcp1 and Tnfα expression, and 16:1n7 promoted a more marked increase in it. In summary, the data show that palmitoleic acid promotes metabolic changes and partially prevents the increase in gene expression on adipocytes triggered by obesity, suggesting that HFD+n7 animals do not require the same magnitude of metabolic adaptation to cope with energy demand from the HFD. In the long term, the effects of 16:1n7 may be more evident and beneficial for the function/dysfunction of WAT from an obese organism, with relevant repercussions in the systemic metabolic homeostasis.