Poxviruses are an important family of viruses with members capable of infecting humans including variola virus, the causative agent of smallpox. Homologs of the variola virus B22 protein are found in almost all chordopoxviruses and have estimated masses of greater than 200 kDa, making them the largest known poxvirus proteins, and leading to our initial interest in the role of this protein family. Most homologs are found in the variable region of the poxviral genome where less conserved host-interaction proteins are found, however, B22 homologs are remarkably well conserved, missing only from the parapox subfamily and vaccinia virus, and sometimes even found in multiples (crocodilepox, canarypox) despite their large size. We showed that the cowpox homolog, CPXV219, was expressed early during infection and cleaved into fragments that remained associated. CPXV219 was observed in the secretory pathway and at the plasma membrane with the majority of the protein extracellular. However, mutants that did not express CPXV219 replicated normally in cell culture and retained virulence in a mouse respiratory infection model. Next, we investigated the importance of the ectromelia virus homolog C15 in the natural infection model, mousepox. In the absence of C15 following footpad infection of BALB/c mice, there was less mortality and at high doses all mice survived, whereas none survived infection with wildtype virus. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less virus in popliteal and inguinal lymph nodes, spleen and liver, key organs in mousepox disease, indicating decreased virus spread and replication in the absence of C15. Decreased spread was not the result of an intrinsic viral function of C15 as the survival of infected mice was dependent on host CD4+ and CD8+ T cells during infection. These results highlight the importance of choosing an appropriate animal model. The influence of B22 homologs displays a species-specificity as observed in the natural virus-host model of ectromelia virus, which provides an excellent model for the study of such host-defense molecules during poxvirus infection. Future work should focus on determining the mechanism of action of these homologs.