Human coxsackievirus B3 (CVB3) is known to trigger in host cells a biphasic activation of extracellular signal-regulated kinase (ERK1/2); i.e., early transient and late sustained activation. In this study, we explored (i) the role of ERK1/2 activation in virus entry into cells and virus replication and (ii) cellular genes influenced by this activation in CVB3-infected HeLa cells. Pretreatment of the cells with an ERK1/2 inhibitor, PD98059 showed that early transient ERK1/2 activation is not be related to virus entry, but late sustained ERK1/2 activation plays a role in virus replication. To identify which cellular genes are influenced by the ERK1/2 activation after virus infection, a cDNA microarray analysis was performed. In HeLa cells pretreated with PD98059 and then infected with the virus, the number of influenced cellular genes was higher compared to that in infected cells not pretreated with the inhibitor (15 vs 77 at 10 mins post infection (p.i.) and 347 vs 91 at 9 hrs p.i. Thus the virus infection affected several host genes through ERK1/2 activation.