Vancomycin (VCM) is an essential glycopeptide antibiotic employed for treating methicillin-resistant Staphylococcus infections. However, its clinical use is limited by nephrotoxicity. Vitamin B12 (Vit B12) possesses antioxidant, anti-inflammatory, and anti-fibrotic properties that may protect against nephrotoxicity. However, Vitamin B12 bioavailability is inherently low. Therefore, nanotechnology-based approaches have been employed to overcome these limitations. Our research examined the formulation and preclinical assessment of Vitamin B12 nanocapsules (Vit B12 NC) against VCM-induced oxidative and, endoplasmic reticulum (ER) stress, inflammation, and fibrosis in rats. Nephrotoxicity was induced by administering VCM, followed by treatment with oral Vit B12 NC. Renal function, oxidative and ER stress markers, inflammatory cytokines, fibrosis markers, and histopathological changes in kidney tissue were evaluated. Vit B12 NC treatment reduced serum creatinine, uric acid, and urea levels, raised antioxidant enzyme activities (catalase and total antioxidant capacity), and decreased malondialdehyde (MDA) levels. It also downregulated ER stress markers, including inositol-requiring enzyme 1 (IRE1), TNF receptor-associated factor 2 (TRAF2), c-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP). Inflammatory mediators such as Toll-like receptor 4 (TLR4), interleukin-17 (IL-17), and interleukin-18 (IL-18) were also repressed. Also, it reduced renal fibrosis as indicated by decreased expression of VIM, miR-382-5p, and miR-92a-3p. Furthermore, it reversed the histopathological alterations in renal tissues. These findings suggest that Vit B12 NC exhibits promising nephroprotective potential against VCM-induced nephrotoxicity.