Background: Penicillin belongs to the β-Lactam group of antibiotics which is used as a first-line drug in veterinary medicine. The occurrence of penicillin anaphylaxis and its pathophysiology is rarely reported in ruminants. Methods: In the present study, oxidative stress and pro-inflammatory response were investigated in 4 cases (Jersey crossbred) of penicillin hypersensitivity by assessing oxidative stress biomarkers and pro-inflammatory cytokines. Animals in the current experimental plan were divided into three groups, group I included penicillin hypersensitive (n=4), group II included penicillin tolerant (n=6) and group III (n=10), included healthy control animals. Result: Mean values of temperature, heart rate and respiration were significantly (p less than 0.05) elevated in the penicillin-sensitive group compared to the penicillin tolerant and control group. The present study revealed significantly (p less than 0.05) elevated levels of lipid peroxidation (MDA), xanthine oxidase (XO), nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), while as levels of glucose-6-phosphate dehydrogenase (G6PD), superoxide dismutase (SOD) and glutathione peroxidase (GPx) were significantly reduced in the penicillin-sensitive group. Levels of G6PD showed significant positive correlation with GPx (r=0.54, P less than 0.001) and catalase (r=0.34, P less than 0.05) while as significant negative correlation with XO (r=0.39, P less than 0.05) was observed. Collectively, these findings indicate that the pathophysiology of penicillin hypersensitivity involves anti-oxidant imbalance and enhanced pro-inflammatory response and targeting these pathways can have therapeutic implications for penicillin hypersensitivity.