Oxidized linoleic acid metabolites (OXLAM) are products of adipocyte lipolysis with the potential to modulate adipose tissue (AT) lipid metabolism and inflammation. In periparturient cows, linoleic acid is preferentially mobilized from AT by hormone-sensitive lipase (HSL) during lipolysis compared with other polyunsaturated fatty acids. Enzymatic and non-enzymatic reactions generate OXLAM. Among OXLAM, 9, 10, and 12- hydroxy-octadecadienoic acids (HODE) have pro- inflammatory functions whereas 9- and 13-oxo- octadecadienoic acids (oxoODE) and 13-HODE can facilitate anti-inflammatory responses and promote lipogenesis. High 9- HODE:9-oxoODE (R1) and 13-HODE:13-oxoODE (R2) ratios are associated with dysregulated inflammation. This study evaluated the effect of HSL activity on OXLAM biosynthesis by subcutaneous AT explants collected from multiparous dairy cows (n = 3) at 10 d before and 10 and 24 d after calving. Explants were treated for 3 h with the β adrenergic agonist isoproterenol (CON 0M; ISO, 10⁻⁶M) to induce HSL activity. HSL contribution to OXLAM biosynthesis was determined by inhibiting its activity with CAY10499 (10⁻⁴M) in the absence (CAY) or presence (CAYISO) of adrenergic stimulation. After treatments, media and explants were collected for lipidomic analysis using HPLC- MS/MS. The statistical model included animal as random effect and the fixed effects of treatment, time relative to calving, and their interaction. ISO increased the biosynthesis of 9-, 12-, and 13-HODE and 9- and 13-oxoODE, and reduced R1 and R2 compared with CON and CAY (P < 0.05). CAYISO partially reversed ISO effects on HODE and 13-oxoODE (P < 0.05); however, 9-oxoODE was not reduced by CAYISO (P = 0.26). The biosynthesis of 12-, 13-HODE and 13-oxoODE was higher and R1 and R2 were lower in ISO at −10d compared with 10d and 24d (P < 0.05). Our ex-vivo model demonstrates for the first time a direct effect of HSL activity on the biosynthesis of OXLAM in AT especially 10 d before parturition. However, biosynthesis of anti-inflammatory OXLAM is limited postpartum and may promote AT inflammation and lipolytic responses to negative energy balance.