Although Mycoplasma bovis, an emerging etiological agent of bovine mastitis, lack classical virulence factors, they induce an immune reaction and inflammation in the host. Because effects on the bovine mammary immune system are not yet well characterized, this study aimed to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC in 4th passage of 4 different cows were challenged with live or heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis in Switzerland with a multiplicity of infection (MOI) of 108, as well as with the type strain PG45 with a MOI of 30. The immune response of bMEC was evaluated after a co-incubation with mycoplasmas for 6, and 24 h at 37°C by measuring the relative mRNA expression of important immune factors by quantitative PCR. Live JF4278 M. bovis triggered a considerable immune response in bMEC (P < 0.05), reflected by the upregulation of relative mRNA expression (ΔΔCT) of tumor necrosis factor (TNF)-α (6 h: 4.5 ± 0.9; 24 h: 4.9 ± 0.4 threshold cycles [CT]), interleukin(IL)-1β (6 h: 7.9 ± 1.0; 24 h: 8.1 ± 0.7 CT), IL-6 (24h: 2.1 ± 0.4), IL-8 (6 h: 5.6 ± 0.9; 24 h: 7.1 ± 1.1 CT), lactoferrin (after 24 h: 4.4 ± 1.0 CT), Toll-like receptor(TLR)-2 (6 h: 1.9 ± 0.3 CT), and serum amyloid A (SAA; 24 h: 8.5 ± 0.7 CT). For live type strain PG45, very similar results were obtained; that is, a significant induction of TNF-α (6 h: 4.9 ± 0.4; 24 h: 4.4 0.5 CT), IL-1β (6 h: 7.9 ± 1.1; 24 h: 9.2 ± 0.5 CT), IL-8 (6 h: 5.6 ± 0.3; 24 h: 7.6 ± 1.0 CT), lactoferrin (6 h: 1.1 ± 0.2; 24 h: 4.0 ± 1.4 CT), TLR-2 (24 h: 2.3 ± 0.7 CT), and SAA (24 h: 8.5 ± 0.7 CT). Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis may indicate that its intracellular localization or secondary metabolites are involved in M. bovis pathogenesis.