Objective - To determine the anti-inflammatory effects of glycyrrhizin (GL) in lactating cows with mastitis attributable to naturally occurring infection with coagulase-negative staphylococci (CNS). Animals - 12 lactating Holstein cows with mastitis attributable to infection with CNS and 2 healthy cows without mastitis. Procedure - Clinical signs, number of bacteria in milk, somatic cell count (SCC) in milk, concentrations of α-lactalbumin and lactoferrin in milk, and concentration of histamine in milk were investigated before and after intramammary infusion of GL (6 cows) or antimicrobials (6 cows). Glands of 2 healthy cows were infused with staphylococcal enterotoxin; milk leukocytes were then harvested and incubated with various doses of GL. Results - In cows infected with CNS that had a low bacterial concentration in milk, infusion of GL alone resulted in significant improvements in swelling, firmness of glands, and number of clots in milk, and it decreased the SCC, but not significantly. Percentage of neutrophils decreased significantly (to < 30%) by 2 days after infusion. Use of lactoferrin as a marker of inflammation in mammary glands revealed a decrease in concentrations, whereas use of α-lactalbumin as a marker of recovery for mammary glands revealed significant increases in concentrations in the GL-infused group. Accompanying these anti-inflammatory effects, a decrease in the concentration of histamine in milk was observed in the GL-infused group. Glycyrrhizin decreased histamine production by milk leukocytes in a concentration-dependent manner. Conclusions and Clinical Relevance - Infusion of GL may regulate intramammary inflammation through modulation of inflammatory mediators such as histamine.