Periparturient dairy cows with high body condition score (BCS) exhibit enhanced adipose tissue (AT) lipolysis that promotes uncontrolled inflammatory responses. Among the fatty acids (FA) released during lipolysis, polyunsaturated (PUFA) modulate inflammation through their oxidized byproducts (oxylipids). Linoleic acid (LA) and arachidonic acid (ArA) derived oxylipids, hydroxy-octadecadienoic acids (HODE), and hydroxy-eicosatetraenoic acids (HETE) act as pro-inflammatory mediators, while oxylipids from eicosapentanoic acid (EPA) are anti-inflammatory. Currently, there is minimal information available on the effect of lipolysis and BCS on oxylipid biosynthesis in dairy cows. We hypothesized that periparturient PUFA and oxylipid profiles are dependent on BCS and lipolysis intensity. Holstein cows with high (HB; BCS ≥ 3.75, n = 5) or moderate (MB; BCS ≤ 3.5, n = 4) BCS were selected at dry-off. Blood and subcutaneous AT samples were collected at −27 ± 7 (D1) and −10 ± 5 d (D2) prepartum and at 8 ± 3 d postpartum (PP). Targeted lipidomic analysis was performed on samples using HPLC-MS/MS. The statistical model included the random effect of block and the fixed effect of treatment, time, and their interaction (analyzed in SAS). Plasma FA concentrations increased as parturition approached peaking at PP. Cows with HB had higher plasma FA compared with MB, reflecting a BCS effect on lipolysis intensity (P < 0.01). Plasma ArA and EPA were decreased at D2, compared with the other time points for all cows (P < 0.01). Cows with HB had lower plasma content of ArA and EPA compared with MB at all time points (P < 0.05). Concentrations of ArA and EPA in AT, as well as LA content in AT and plasma, remained unchanged during the experiment and were not influenced by BCS. In AT, 13-HODE, 5-HETE, and 11-HETE were increased at PP compared with D1 and D2 (P < 0.05). Concentrations of 9-HODE,13-HODE, 5-HETE, and 15-HETE in AT were decreased in HB compared with MB (P < 0.05). Our results demonstrate that prepartum adiposity may limit the availability of plasma PUFA, such as EPA, which serve as substrates for anti-inflammatory oxylipids. Furthermore, periparturient lipolysis enhances HODE and HETE biosynthesis in AT and their release into circulation.