Although gemcitabine is a primary chemotherapy for pancreatic cancer, its effectiveness is limited by chemoresistance and nephrotoxicity, posing significant clinical challenges. Therefore, the development of novel therapeutic approaches to prevent pancreatic malignancy remains crucial. This study aimed to investigate the potential of melatonin in enhancing gemcitabine's anticancer efficacy while mitigating its nephrotoxic effects through modulation of the Keap1/p62 pathway. A pancreatic cancer xenograft model was established in rats, which received either gemcitabine (50 mg/kg, I.P.), melatonin (50 mg/kg, I.P.), or their combination three times per week for 2 weeks. Our findings demonstrate that melatonin potentiates gemcitabine's cancer-suppressing effects via modulation of the Kelch-like-ECH associated protein-1 (Keap1)/p62 pathway, resulting in reduced fibrosis, oxidative stress, and inflammatory markers. Additionally, melatonin significantly mitigated gemcitabine-induced nephrotoxicity. These results suggest that melatonin may serve as an adjuvant therapy in pancreatic cancer treatment, enhancing chemotherapy efficacy while reducing its adverse effects.