The aim of the present study was to assess omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in liver tissue and evaluate changes in the n‑6-associated inflammatory pathway following liver ischemia/re‑perfusion (IR) injury. Male Wistar rats which were allowed free access to standard rat chow were included in the study. Blood vessels supplying the median and left lateral hepatic lobes were occluded with an arterial clamp for 60 min, followed by 60 min of re‑perfusion. Levels of arachidonic acid (AA, C20:4n‑6), dihomo‑gamma‑linolenic acid (DGLA, C20:3n‑6), eicosapentaenoic acid (EPA, C20:5n‑3) and docosahexaenoic acid (DHA, C22:6n‑3) in liver tissue were determined by an optimized multiple reaction monitoring method using ultra fast‑liquid chromatography coupled with tandem mass spectrometry. Phospholipase A2 (PLA2), cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in tissue samples to evaluate changes in the n‑6 inflammatory pathway. Total histopathological score of cellular damage were significantly increased following hepatic IR injury. n‑3 and n‑6 PUFA levels were significantly increased in post‑ischemic liver tissue compared to those in non‑ischemic controls. No significant difference was observed in the AA/DHA and AA/EPA ratio in post‑ischemic liver tissues compared with that in the control. Tissue activity of PLA2 and COX as well as PGE2 levels were significantly increased in post‑ischemic liver tissues compared to those in non‑ischemic controls. The results of the present study suggested that increased hydrolysis of fatty acids via PLA2 triggers the activity of COX and leads to increased PGE2 levels. Future studies evaluating agents which block the formation of eicosanoids derived from n‑6 PUFAs may facilitate the development and application of treatment strategies in liver injury following IR.