Objective— miR-33 has emerged as an important regulator of lipid homeostasis. Inhibition of miR-33 has been demonstrated as protective against atherosclerosis; however, recent studies in mice suggest that miR-33 inhibition may have adverse effects on lipid and insulin metabolism. Given the therapeutic interest in miR-33 inhibitors for treating atherosclerosis, we sought to test whether pharmacologically inhibiting miR-33 at atheroprotective doses affected metabolic parameters in a mouse model of diet-induced obesity. Approach and Results— High-fat diet (HFD) feeding in conjunction with treatment of male mice with 10 mg/kg control anti-miR or anti-miR33 inhibitors for 20 weeks promoted equivalent weight gain in all groups. miR-33 inhibitors increased plasma total cholesterol and decreased serum triglycerides compared with control anti-miR, but not compared with PBS-treated mice. Metrics of insulin resistance were not altered in anti-miR33–treated mice compared with controls; however, respiratory exchange ratio was decreased in anti-miR33–treated mice. Hepatic expression of miR-33 targets Abca1 and Hadhb were derepressed on miR-33 inhibition. In contrast, protein levels of putative miR-33 target gene SREBP-1 or its downstream targets genes Fasn and Acc were not altered in anti-miR33–treated mice, and hepatic lipid accumulation did not differ between groups. In the adipose tissue, anti-miR33 treatment increased Ampk gene expression and markers of M2 macrophage polarization. Conclusions— We demonstrate in a mouse model of diet-induced obesity that therapeutic silencing of miR-33 may promote whole-body oxidative metabolism but does not affect metabolic dysregulation. This suggests that pharmacological inhibition of miR-33 at doses known to reduce atherosclerosis may be a safe future therapeutic.