We have previously shown that avian reovirus (ARV) S1133 and its structural protein sigmaC cause apoptosis in cultured Vero cells through an unknown intracellular signaling pathway. This work investigates how ARV S1133 induces proapoptotic signals. Upon ARV S1133 infection and subsequent apoptosis, levels of p53 mRNA and protein, and p53 serine-46 and serine-392 phosphorylation increased. In addition, p53-driven reporter activity and levels of the p53-induced apoptotic protein bax were increased, and Src tyrosine-418 phosphorylation was elevated. UV-inactivated virus failed to activate Src, p53 or induce apoptosis. Over-expression of dominant negative p53, or treatment with tyrosine kinase inhibitor genistein protected cells from ARV S1133-induced apoptosis. Inhibition of Src by over-expression of C-terminal Src kinase (Csk) or treatment with Src family tyrosine kinase inhibitor SU-6656 diminished the ARV S1133-induced p53 expression, activation, and apoptosis. Over-expression of sigmaC resulted in the upregulation of p53, p53 serine-46 phosphorylation, p53-driven reporter activity and accumulation of bax. sigmaC expression during ARV S1133 infection was concomitant with the onset of apoptosis. These studies provide strong evidence that the viral gene expression is required for ARV S1133 to initiate a proapoptotic signal via Src to p53. In addition, sigmaC was able to utilize a p53-dependent pathway to elicit apoptosis.