Despite evidence supporting an association between enterovirus (EV) infection and type 1 diabetes, the etiological mechanism(s) for EV-induced beta cell destruction are not well understood. We and others have shown that infection with multiple EVs particularly Coxsackievirus B (CVBs) can induce β-cell production of proinflammatory cytokines and chemokines. In this study we examined pathways of β-cell death following EV infection. We investigated the effects of CVB4 & CVB5 (GenBank accession numbers GQ126859 & GQ126860) on viral replication and expression of factors representing multiple apoptotic pathways (Akt, c-Jun, ERK1/2, JNK, NF-κB, p38 MAPK, p53 & STAT3) in human islet progenitor cells (HIPCs) and the rodent β-cell line INS-1. Viral RNA was determined by quantitative RT-PCR and phosphoprotein levels of apoptotic factors by Bioplex in the infected cells at 4 hrs, 12 hrs, day 1, 2, 3, 4 and 5 post inoculation. Both CVB4 and CVB5 replicated in INS-1 and CVB5 in HIPC at a multiplicity of infection (MOI) of 1 10. CVB5 infection of HIPCs induced JNK, p38 MAPK, ERK1/2 and c-Jun on day 1 post-infection (p<0.05). These factors were also induced in INS-1 cells infected with CVB4 and CVB5 on day 1-2 except for ERK1/2 which increased 4 hours post-infection (p<0.05). NF-κB and STAT3 phosphoprotein increased in INS-1 cells infected with CVB4 and CVB5 on day 1-3 post-infection (p<0.05). However there was no increase in these two phosphoproteins in the HIPC infected with CVB5. Akt which is an anti-apoptotic factor decreased in INS-1 but increased slightly in the HIPC. There were no changes in p53 phosphoprotein levels in INS-1 but it decreased in the HIPC. In summary, we have shown for the first time that CVB4 and CVB5 induce multiple apoptotic pathways in HIPC and INS-1 cells.