Deficiency of serum levels of 25-hydroxyvitamin D(3) has been correlated with increased risk of infectious diseases such as tuberculosis and influenza. A plausible reason for this association is that expression of genes encoding important antimicrobial proteins depends on concentrations of 1,25-dihydroxyvitamin D(3) produced by activated immune cells at sites of infection, and that synthesis of 1,25-dihydroxyvitamin D(3) is dependent on the availability of 25-hydroxyvitamin D(3). Thus, increasing the availability of 25(OH)D(3) for immune cell synthesis of 1,25-dihydroxyvitamin D(3) at sites of infection has been hypothesized to aid in clearance of the infection. This report details the treatment of an acute intramammary infection with infusion of 25-hydroxyvitamin D(3) to the site of infection. Ten lactating cows were infected with in one quarter of their mammary glands. Half of the animals were treated intramammary with 25-hydroxyvitamin D(3). The 25-hydroxyvitamin D(3) treated animal showed significantly lower bacterial counts in milk and showed reduced symptomatic affects of the mastitis. It is significant that treatment with 25-hydroxyvitamin D(3) reduced the severity of an acute bacterial infection. This finding suggested a significant non-antibiotic complimentary role for 25-hydroxyvitamin D(3) in the treatment of infections in compartments naturally low in 25-hydroxyvitamin D(3) such as the mammary gland and by extension, possibly upper respiratory tract infections.