Our objective was to determine if palmitoleic (C16:1 cis-9) acid supplementation to primary bovine adipocytes regulates lipogenic gene expression and rates of lipogenesis. Stromal vascular cells were isolated from subcutaneous and intermuscular fat, propagated, and frozen for use in this study. Cells were passaged 4 times, allowed to reach confluence, held for 2 d, and then differentiated with a standard hormone cocktail (d 0). At d 2, secondary differentiation media containing 1 of 4 concentrations of palmitoleic acid (0, 50, 150, or 300 μM) were added for 10 d. Cells were harvested on d 6 and 12 to assess fatty acid concentrations and gene expression. In addition, (13)C2 and (13)C18:0 stable isotopes were added on d 6 to measure lipogenesis and desaturase activity, respectively. Concentrations of C16:1 and total fatty acids increased (P < 0.05) linearly in response to palmitoleic acid supplement. Concentrations of C18:1 cis-11 and C20:1 cis-13 also increased (P < 0.01) in response to supplementation, suggesting elongation of palmitoleic acid in vitro. Concentrations of C16:1, C18:1 cis-11, and total fatty acids were also greater (P < 0.05) at d 12 compared with d 6. In contrast, C16:0, C18:0, and C18:1 cis-9 concentrations decreased (P < 0.05) in response to palmitoleic acid supplementation and were not affected (P > 0.05) by harvest day. The ratio of C18:1 cis-9/C18:0 and fractional synthetic rate (FSR) of desaturation decreased (P < 0.05) in response to increasing palmitoleic acid supplementation. In addition, FSR of lipogenesis was reduced (P < 0.05) in palmitoleic acid-treated cells. Messenger RNA abundance as determined by real-time quantitative PCR for stearoyl-CoA desaturase 1 (SCD1), fatty acid synthase (FASN), and elongase protein 6 (ELOVL6) genes were reduced (P < 0.05) by palmitoleic acid supplementation. Expression of a β-oxidation gene, carnitine palmitoyltransferase 1A (CPT1A), was upregulated (P < 0.05) with palmitoleic acid supplementation in a dose-responsive manner. Supplementation of palmitoleic acid to bovine adipocytes results in increased incorporation of this fatty acid and its elongation products into the adipocyte, which downregulates SCD1, FASN, and ELOVL6 to decrease lipogenesis and upregulates CPT1A, potentially increasing β-oxidation. These results suggest that palmitoleic acid, an end product of desaturation, can act as a regulator of lipogenesis, desaturation, and β-oxidation in bovine adipocytes.