Hydropericardium-Hepatitis Syndrome (HHS) caused by Fowl Adenovirus Serotype 4 (FAdV4) infection is a severe threat to the poultry industry worldwide, especially in China since 2015. Recent studies show that FAdV4 induces liver injury through apoptosis. However, the underlying molecular mechanism is still unclear. We report here that FAdV4 infection caused apoptosis in Leghorn male hepatocellular (LMH) cells and that PX, a structural protein of FAdV4, acted as a major viral factor inducing apoptosis. Furthermore, the nuclear localization of PX is determined by the R/K regions of PX and required for PX-induced apoptosis. Moreover, alanines 11 and 129 of PX are crucial to PX-induced apoptosis. Inhibition of FAdV4-induced apoptosis by caspase inhibitors retarded viral replication, suggesting that PX serves as a virulence factor for FAdV4 infection, which may further our understandings of the pathogenesis of FAdV4 infection.