Mycoplasma canis is an opportunistic pathogen of dogs, isolated from dogs with urogenital diseases and infertility. It has also been isolated from other mammals. Experimental infections with M. canis produced chronic urethritis, epididymitis and endometritis. However, more work is needed to establish whether M. canis is linked to infertility. Little is known about virulence factors and pathogenicity of canine mycoplasmas and their effect on the host. We analyzed the influence of M. canis infection on the immortilized canine cell line DH82. After infection of the DH82 cell line with M. canis we followed the changes in viability and morphology of DH82 cells. Using the real-time PCR method, we analyzed relative changes in gene expression of inflammatory cytokines IL-1β, IL-6, IL-8 and transcription factor NFĸB1. Moreover, we analyzed the release of nitric oxide (NO) and the concentration of viable bacteria in DH82 cell supenatants during infection. The infection resulted in gradual increase of IL-1β gene expression. The expression of IL-6 gene gradually increased until 6 h after infection and dropped until 12 h after infection. Also IL-8 gene expression gradually increased during infection, however the expression was the highest 48 h after the infection. The viability of DH82 cells dropped 24 h and even more obviously 48 h after infection. The concentration of viable bacteria in DH82 cell supernatants varied during the infection and it was the lowest 48 h after the infection. We did not detect any changes in NO concentrations in DH82 cell supernatants after the infection. The gained results give us a partial insight into the M. canis infection of the eukaryotic cells.