BackgroundObesity is the leading chronic disease affecting people of all ages. The objective of this study was to optimize composition of a bitter melon seed oil (BMSO) product to maximize its anti-adiposity effect.MethodsBleaching oil, saponifiables and non-saponifiables were prepared from BMSO, with α-eleostearic acid (α-ESA) content in BMSO maintained in bleaching oil and saponifiables. C57BL/6 J mice were allocated into five groups (n = 10/group) to receive diet C [30% soybean oil (SBO)], BM [25% SBO + 5% BMSO], BMS, BMNS or BMD. For the three latter diets, saponifiables (hydrolyzed fatty acids from BMSO), non-saponifiables (excluding fatty acids from BMSO) or bleaching oil (excluding pigments from BMSO), respectively, were added in amount equivalent to their content in 5% BMSO and SBO was added to bring total fat to 30%. After 14 wk., indices associated with adiposity and safety, as well as lipid metabolic signaling in white adipose tissue (WAT), were measured.ResultsThe body fat percentage of mice in group BM, BMS, BMNS, and BMD were 90 ± 26, 76 ± 21, 115 ± 30 and 95 ± 17% of that in group C. Based on body fat percentage and plasma leptin concentrations, an anti-adiposity effect was evident in groups BM, BMS and BMD (greatest effect in BMS). Histologically, inguinal fat had smaller adipocytes in groups BM, BMS and BMD (P < 0.05), but not in group BMNS, relative to group C. There were no differences among groups in blood pressure or heart rate. Moreover, Sirt1 mRNA levels in inguinal fat were significantly greater in groups BM, BMS and BMD than group C.ConclusionWe concluded that the anti-adiposity function of BMSO was solely attributed to the fatty acid fraction, with the free fatty acid form having the greatest effect.Electronic supplementary materialThe online version of this article (10.1186/s12944-017-0578-3) contains supplementary material, which is available to authorized users.