Background Prolactin (PRL) has been reported to be associated with oxidative stress, which is an important contributor leading to cell apoptosis. However, little is known about the mechanisms underlying the effects of PRL on cytotoxicity and oxidative stress in ovine ovarian granulosa cells (GCs). Methods Ovine ovarian GCs were treated with 0, 4, 20, 100 and 500 ng/mL of PRL. Then, the cytotoxicity, cell viability, malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of GCs were detected. Additionally, 500 ng/mL PRL was chosen as the high PRL concentration (HPC) due to its high cytotoxicity and oxidative stress. Proteomic and metabonomic were performed to examine the overall difference in proteins and metabolic pathways between C (control: 0 ng/mL PRL) and P groups (500 ng/mL PRL). Results The results indicated that GCs treated with 4 ng/mL PRL significantly decreased (P