We previously reported that postpartum supplementation of oleic acid (cis-9 C18:1; OA) increased milk yield and reduced plasma NEFA and body weight loss; however, the mechanisms driving these responses are unknown. In rodent adipose tissue (AT), OA enhances lipogenesis and potentiates the effects of insulin; however, responses to OA supplementation in bovine AT have not been characterized. Our objective was to determine the effects of postpartum OA supplementation on AT responses to isoproterenol and insulin. Multiparous cows were infused abomasally with 60 g/d of OA (n = 6) or vehicle (VEH, n = 6) from 1 to 15 d postpartum. Subcutaneous AT (SCAT) explants were collected at −14, +6, and +12 d relative to parturition and incubated for 3 h at 37°C with 0 µM (CON) or 1 µM of β adrenergic agonist isoproterenol (ISO) to induce lipolysis. The anti-lipolytic effect of insulin at low (0.2 µg/L, INL) and high concentration (1 µg/L, INH) was determined during ISO stimulation. Lipolytic responses were evaluated by quantification of glycerol release. ISO responses are reported as % increase over CON. Insulin responses are reported as % decrease over ISO. Statistical analysis was performed using a mixed linear model, considering as fixed effects OA infusion, time, treatments and their interaction. Across treatments and sample time points, ISO increased the lipolytic responses by 300 ± 25% compared with CON (P < 0.001). Lipolytic response to ISO was not different between VEH and OA groups before infusions (−14 d). Compared with CON, OA infusion reduced lipolytic response to ISO at +6 d (VEH = 177 ± 6.5% and OA = 167 ± 6.5%; P = 0.06) and at +12 d (VEH = 258 ± 7.8% and OA = 157 ± 7.8%; P = 0.02). Compared with ISO, INL and INH inhibited lipolysis by −170 ± 2.5% and −231 ± 2.5%, respectively (P < 0.001). The anti-lipolytic effect of INL and INH did not differ between VEH or OA groups before infusions (−14 d). At +6 and +12 d, INH had a stronger anti-lipolytic response in OA (−222 ± 5.5% and −238 ± 7.2%) compared with VEH (−106 ± 5.5% and −128 ± 7.2%; P < 0.05), respectively. Results demonstrate that oleic acid supplementation immediately postpartum reduces lipolytic responses and improves insulin sensitivity of AT in early lactation dairy cows.