In non-ruminants, the sphingolipid ceramide reduces insulin sensitivity by inactivating protein kinase B (AKT) within the insulin signaling pathway. We have established that ceramide accumulation develops with impaired systemic insulin action in ruminants during the transition from gestation to lactation, dietary palmitic acid supplementation, controlled nutrient restriction, or intravenous triacylglycerol infusion. We hypothesized that ceramide promotes AKT inactivation and antagonizes insulin sensitivity in primary bovine adipocytes. Stromal-vascular cells were grown from bovine subcutaneous adipose tissue explants and cultured in differentiation media. To modify ceramide supply, we treated differentiated adipocytes with an inhibitor of de novo ceramide synthesis (10 µM myriocin) or cell-permeable C2:0-ceramide (100 µM) for 18 or 2 h, respectively. Untreated controls were included for comparison. Insulin-stimulated AKT activation (i.e., Ser-473 phosphorylation) and 2-deoxy-D-[3H]-glucose (2DOG) uptake were measured using immunoblotting and radioactivity assays, respectively. Adipocyte ceramide concentrations were measured using LC/MS. Data were analyzed under a mixed model including the fixed effect of treatment and the random effect of experiment and replicate within treatment. Relative to undifferentiated adipocytes, triacylglycerol accumulation was ~7-fold greater post differentiation with visible lipid droplet formation (P < 0.01). Pronounced reductions in total ceramide, monohexosylceramide, and lactosylceramide concentrations were observed in differentiated adipocytes treated with myriocin (P < 0.01). For example, myriocin decreased C22:0 and C24:0 ceramide by ~77% (P < 0.01). The insulin-stimulated ratio of phosphorylated AKT to total AKT increased with myriocin by 190% (β-actin normalized; P < 0.05), whereas the ratio of phosphorylated AKT to total AKT decreased by 76% with C2:0-ceramide (P < 0.05). Moreover, adipocyte insulin-stimulated 2DOG uptake was decreased with C2:0-ceramide and increased with myriocin (P < 0.05). We conclude that ceramide inhibits insulin stimulated glucose uptake by downregulating AKT activation in primary bovine adipocytes.