AbstractCurrent vaccines to Escherichia coli mastitis have shown some albeit limited efficacy. Their mode of action has not been documented, and immune responses protecting the mammary gland against E. coli are not completely understood. To improve our knowledge of mammary gland immune protection, cows immunized either intramuscularly or intramammarily with the E. coli P4 were submitted to a homologous mastitis challenge. A third group of mock-immunized cows serve as challenge controls. Local immunization modified favorably the course of infection, by improving bacterial clearance while limiting inflammation. Systemic clinical signs and reduction in milk secretion were also contained. This occurred with a modification of the cytokine profile, such as an increase in IFN-γ and a reduction in TNF-α concentrations in milk. Concentrations of IL-17A and IL-22 increased in milk at the onset of the inflammatory response and remained high up to the elimination of bacteria, but concentrations did not differ between groups. Accelerated bacteriological cure was not linked to an increase in the initial efficiency of phagocytosis in milk. Results support the idea that antibodies did not play a major role in the improvement, and that cell-mediated immunity is the key to understanding E. coli vaccine-induced protection of the mammary gland.