The aim of this study was to determine how hormone-sensitive lipase (HSL) contributes to adipose lipolysis and how insulin regulates lipolysis in periparturient dairy cows. Subcutaneous adipose tissue (SCAT) samples were taken from multiparous dairy cows (n = 22) at 10 d prepartum (dry) and 10 d (fresh) and 24 d (lactation) postpartum. Adipose lipolysis was determined using a short-term in vitro explant culture (3h). Basal lipolysis was determined without addition of reagents. Stimulated lipolysis was determined by β-adrenergic stimulation with isoproterenol (ISO, 10⁻⁶ M). The inhibitory effect of insulin (1 µg/L) was determined on stimulated lipolysis. HSL contribution to basal lipolysis was determined by adding an HSL inhibitor, CAY10499 (CAY, 2 µM). Statistical analyses were performed in R using a mixed effect linear model. Basal lipolysis was higher in SCAT explants from dry cows (1,295 ± 120 nmol glycerol/10⁶ adipocytes) compared with fresh cows (661 ± 114 nmol glycerol/10⁶ adipocytes, P < 0.05). Inhibition of basal lipolysis by CAY was negligible in dry cows (−3.54 ± 5.05% of basal glycerol release), while in fresh and early lactation cows, CAY inhibited basal lipolysis by 36.05 ± 4.51% and 43.05 ± 4.83%, respectively (P < 0.05). ISO stimulated lipolysis in SCAT explants was not different across periods (P > 0.1). Inhibition of stimulated lipolysis by insulin was more pronounced in the dry period (−23.23 ± 3.45%) compared with the fresh period (−9.64 ± 3.24, P < 0.05). Explants with larger adipocytes had higher basal lipolysis (P < 0.001) while adipocyte size did not influenced lipolysis in explants cultured with CAY, ISO or insulin (P > 0.1). Our results demonstrate that the contribution of HSL to basal lipolysis is negligible in the dry period; however, HSL is the major driver of lipolytic responses in SCAT postpartum. Lower basal lipolysis in early lactation suggests that reduced lipogenesis is an important contributor to early lactation fatty acid release from SCAT. Loss of adipocyte sensitivity to the anti-lipolytic action of insulin develops in the early lactation period, which is suggestive for insulin resistance of the lipolytic activity.