The anti-lipogenic effects of trans-10,cis-12 conjugated linoleic acid (T10C12) are well characterized in monogastric species. Similarly, the impact of abomasal infusion of T10C12 on milk fat synthesis in lactating dairy cows is well established. However, the effect of T10C12 on the lipogenic capacity of bovine adipocytes from omental (OM) and subcutaneous adipose tissues (AT) of lactating dairy cows is unknown. Our objective was to evaluate the effects of T10C12 on the lipogenic activity of 3D cultured bovine adipocytes. OM and tailhead (TH) AT samples were collected from 5 lactating non-gestating mature Holstein dairy cows. AT samples were digested with collagenase type II to harvest stromal vascular cells. Preadipocytes were selected by outgrowth of plastic adherent cells and then seeded 3 dimensionally using collagen type I gels. Cells were induced to differentiate using standard adipogenic medium for 14 d. During induction, adipocytes were supplemented with 50 μM T10C12 or 50 μM cis-9,trans-11 (C9T11) using fatty acid free bovine serum albumin (BSA) as carrier. Triglyceride (TAG) accumulation was evaluated quantitatively (unit = µM/µg DNA) using a fluorometric adipogenic kit. Statistical analysis was performed using linear mixed models. Independent of treatment, adipocytes from TH accumulated more TAG than those from OM (28.3 ± 2.92 vs. 20.2 ± 2.92; P < 0.01). Across sites, adipocytes treated with T10C12 had less TAG (18.1 ± 3.21) than those exposed to BSA (25.8 ± 3.21) or C9T11 (28.8 ± 3.21; P < 0.05). TH adipocytes treated with C9T11 accumulated more (P < 0.05) TAG (35.17 ± 4.12) than those exposed to BSA (27.9 ± 4.12). In turn, T10C12 treated TH adipocytes had lower TAG content (21.9 ± 4.12) than cells that were BSA treated. OM adipocytes exposed to T10C12 (14.3 ± 3.71) had lower TAG compared with those treated with C9T11 (22.3 ± 3.71) and BSA (23.9 ± 3.71). Results suggests that T10C12 has a marked anti-lipogenic effect in bovine adipocytes from OM and TH, similar to that observed in monogastric species, and that C9T11 may enhance lipogenesis in the subcutaneous TH AT depot of lactating dairy cows.