Abdominal obesity is prevalent in women and during menopause, making them more susceptible to weight gain, fat redistribution, and subsequent development of metabolic syndrome and associated diseases such as non-alcoholic fatty liver disease (NAFLD). Evidence from menopausal/postmenopausal women has demonstrated an association between declining oestrogen (i.e. 17β oestradiol; E2) levels and the development/progression of both obesity and associated diseases. Furthermore, dietary intake of the micronutrient selenium (Se) is reduced in obese postmenopausal women and a negative correlation between Se level and body mass index (BMI) has been reported. This suggests that novel nutritional and hormonal solutions are needed to moderate fat deposition in postmenopausal women. This study used mouse 3T3-L1 and human HepG2/C3A cells, as in vitro models of obesity and NAFLD, respectively, to understand basic cellular mechanisms associated with lipogenesis, and to study the role of Se and oestrogen, as E2, in modulating lipid deposition. Supplementation of 3T3-L1 cells during differentiation to adipocytes with 200 nmol/L Se reduced lipid deposition (~20%) by increasing the expression of genes related to redox status (Gpx1, Selenow, and Ucp2) and reducing the expression of markers of energy metabolism, inflammation and adipocyte differentiation (Lep, Cox-2, and Fabp4); whereas administration of 10 nmol/L E2 regulated lipid synthesis and metabolism (reductions in Fasn, Pparg, and Hsl expression and increased Fabp4 and Glut4 expression). In HepG2/C3A cells, both Se and E2 reduced lipid accumulation (15%−20%), via regulation of lipid and energy metabolism and inflammatory genes (SREBF1, SCD1, COX2, and LEP). These results suggest that both hormonal treatment and micronutrient supplementation may be beneficial in obesity and NAFLD management. If our current in vitro findings were subsequently demonstrated in vivo, they could provide valuable data to support the use of either Se supplementation and/or oestrogen-based therapies to prevent and manage obesity and NAFLD in postmenopausal women.