This pioneering study explored the therapeutic potential of human Wharton's jelly derived mesenchymal stem cells (hWJ-MSCs) as a novel regenerative treatment for subclinical (SCM) and clinical mastitis (CM) in dairy cows, a condition that poses significant economic and health challenges. hWJ-MSCs were isolated and characterized, and then administered intramammarily in two doses, seven days apart, to twelve cows that were divided into control, SCM, and CM groups. Comprehensive evaluations, including somatic cell count (SCC), proinflammatory cytokine profiles, milk yield, and bacteriological analyses, were conducted before treatment and on days 14 and 21 after treatment. The results revealed a remarkable reduction in SCC by 29.62 % in the SCM group and 70.46 % in the CM group, accompanied by a pronounced suppression of proinflammatory cytokines such as TNF-α and IL-1β. hWJ-MSCs therapy also led to substantial improvements in milk yield, with increases of 39.38 % and 14.32 % in the SCM and CM groups, respectively. Moreover, bacteriological analyses revealed the complete eradication of pathogenic agents, including Staphylococcus aureus and Escherichia coli, in nearly all treated patients. These findings underscore the potential of hWJ-MSCs to redefine mastitis management by offering a safe, effective, and antibiotic-free alternative, addressing both agricultural and global health concerns related to antimicrobial resistance. The significant therapeutic effects observed in this study suggest that hWJ-MSCs could be used to treat mastitis, paving the way for advanced stem cell-based interventions in veterinary medicine. Further large-scale studies are warranted to validate these promising results and establish hWJ-MSCs as a cornerstone of sustainable livestock health management.