The family Poxviridae comprises the most complex animal DNA viruses. During some poxvirus infections, A-type inclusion bodies (ATIs), codified by the ati gene, are produced. Although some studies have compared poxviruses that encode these inclusion bodies with those that do not, the biological function of ATIs is poorly understood. A recombinant ati-deleted cowpox virus was constructed and compared with the wild-type virus in in vitro experiments including electron microscopy and plaque and viral growth assays. No significant differences were observed in vitro. This reinforces the conclusion that the inclusion body is not essential for in vitro viral replication and morphogenesis. Additionally, different lesion progressions in vivo were observed by macroscopic and histological analysis, suggesting that the presence or absence of ATIs could result in different healing dynamics. This is the first time that the role of ATIs during viral replication has been studied based solely on one variable, the presence or absence of ATIs.