Several mammalian viruses have been shown to induce a cellular DNA damage response during replication, and in some cases, this response is required for optimal virus replication. However, nothing is known about whether a DNA damage response is stimulated by DNA viruses in invertebrates. Cell cycle arrest and apoptosis are two of the downstream effects of the DNA damage response, and both are stimulated by baculovirus infection, suggesting a possible relationship between baculoviruses and the DNA damage response. In the study described in this report, we found that replication of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in the cell line Sf9, derived from the lepidopteran insect Spodoptera frugiperda, stimulated a DNA damage response, as indicated by an increased abundance of the S. frugiperda P53 protein (SfP53) and phosphorylation of the histone variant protein H2AX. Stimulation of the DNA damage response was dependent on viral DNA replication. Inhibition of the DNA damage response prevented both the increase in SfP53 accumulation and H2AX phosphorylation and also caused a 10- to 100-fold reduction in virus production, along with decreased viral DNA replication and late gene expression. However, silencing of Sfp53 expression by RNA interference did not significantly affect AcMNPV replication or induction of apoptosis by a mutant of AcMNPV lacking the antiapoptotic gene p35, indicating that these processes are not dependent on SfP53 in Sf9 cells.