27-Hydroxycholesterol (27HC) is an abundant cholesterol metabolite and has detrimental effects on the cardiovascular system, whereas its impact on adiposity is not well known. In this study, we found that elevations in 27HC cause increased body weight gain in mice fed a high-fat/high-cholesterol diet in an estrogen receptor α-dependent manner. Regardless of diet type, body fat mass was increased by 27HC without changes in food intake or fat absorption. 27HC did not alter energy expenditure in mice fed a normal chow diet and increased visceral white adipose mass by inducing hyperplasia but not hypertrophy. Although 27HC did not augment adipocyte terminal differentiation, it increased the adipose cell population that differentiates to mature adipocytes. RNA sequencing analysis revealed that 27HC treatment of mice fed a normal chow diet induces inflammatory gene sets similar to those seen after high-fat/high-cholesterol diet feeding, whereas there was no overlap in inflammatory gene expression among any other 27HC administration/diet change combination. Histological analysis showed that 27HC treatment increased the number of total and M1-type macrophages in white adipose tissues. Thus, 27HC promotes adiposity by directly affecting white adipose tissues and by increasing adipose inflammatory responses. Lowering serum 27HC levels may lead to an approach targeting cholesterol to prevent diet-induced obesity.