Palmitelaidic (C16:1 trans-9) acid has been suggested to have beneficial effects on human health, including reduced adiposity. Objectives of this research were to quantify the amounts of palmitelaidic acid in beef samples and determine the effect of palmitelaidic acid supplementation on lipogenesis in bovine preadipocytes and adipocytes in vitro. For the first objective, palmitelaidic acid content of LM samples from steers finished on forage or concentrate systems was determined. Palmitelaidic acid in LM samples from forage-finished beef ranged from 10 to 17 mg/100 g of muscle corresponding to 0.52% to 0.65% of total fatty acids. Forage species grazed during finishing, and animal age at harvest also altered palmitelaidic acid concentrations and contents in the LM of forage-finished beef. Palmitelaidic acid concentration of concentrate-finished beef was lower (P < 0.05; 0.25% vs. 0.56%); however, because of increased (P < 0.05) total fatty acid content with concentrate finishing, amount of palmitelaidic acid was similar (P > 0.05) to beef from steers finished on pearl millet and greater (P < 0.05) than those finished on alfalfa. For the second objective, undifferentiated preadipocytes and differentiated adipocytes were supplemented with 0 to 300 μM of palmitelaidic acid. Palmitelaidic acid supplementation reduced (P < 0.05) cell viability of undifferentiated preadipocytes at greater levels (150 and 300 μM) but did not affect (P > 0.05) the viability of differentiated adipocytes. In preadipocytes, palmitelaidic acid increased (P < 0.05) palmitelaidic and trans-11 vaccenic (C18:1 trans-11) acids at high levels of supplementation (300 μM). In adipocytes, palmitelaidic acid supplementation increased (P < 0.05) palmitelaidic acid, trans-11 vaccenic acid, and total fatty acid content. In addition, cis-9, trans-11 CLA also increased (P < 0.05) with palmitelaidic acid supplementation in adipocytes. These results indicate that palmitelaidic acid can be elongated in both preadipocytes and adipocytes and desaturated in adipocytes to generate trans-11 vaccenic acid and cis-9, trans-11 CLA, respectively. Beef products are a source of palmitelaidic acid in the human diet, which can be elongated and desaturated to produce trans-11 vaccenic acid and cis-9, trans-11 CLA.