DNA viruses, like poxviruses, possess a highly stable genome, suggesting adaption of virus particles to specific cell types is not restricted to genomic changes. Cowpox viruses (CPXV) are zoonotic poxviruses with an extraordinary broad host range, demonstrating their adaptive potential in vivo. To elucidate novel adaption mechanisms of poxviruses, we isolated CPXV particles from a rat and passaged them five times in a human (HEp-2) and a rat (Rat-2) cell line. Subsequently, we purified mature virions and compared the proteome of the non-passaged virions and each passage.