Streptococcus uberis is the most common environmental mastitis pathogen causing udder inflammations of different severities in dairy cows. The aim of the study was to investigate if the different clinical outcome of mastitis induced by different strains of S. uberis can be reflected in the mammary immune response. Mammary epithelial cells and somatic milk cells were treated with heat inactivated and living S. uberis of strain A and strain B in vitro. Strain A was repeatedly isolated from a chronically infected quarter during 8 months, and persisted in the quarter despite antibiotic treatment. Strain B caused an acute clinical mastitis and was not further isolated after a single antibiotic treatment. Treatment with Strain B induced a more pronounced increase of mRNA-expression of various immune factors (interleukin-8, interleukin-1beta, RANTES, and lactoferrin) in mammary epithelial cells than strain A. In contrast to mammary epithelial cells the response of removed somatic milk cells showed no differences between the stimulation with two S. uberis strains. Tumor necrosis factor-alpha mRNA expression was not differently induced by the two strains. In conclusion, the characteristics of different severities of mastitis that are induced by different S. uberis strains in vivo can also be reflected at the level of the immune response of the mammary gland in vitro.