Despite evidence supporting an association between enterovirus (EV) infection and type 1 diabetes, the etiological mechanism(s) for EV‐induced beta cell destruction is(are) not well understood. In this study, the effects of Coxsackievirus B (CVB) 1–6 on cell lysis and cytokine/chemokine expression in the insulinoma‐1 (INS‐1) beta cell line were investigated. Cytolysis was assessed using tissue culture infectious dose 50 (TCID₅₀). Quantitative RT‐PCR was used to measure viral RNA and mRNA of cytokines interferon (IFN)‐α, IFN‐β, IFN‐γ, tumor necrosis factor (TNF)‐α, and chemokine (C–X–C motif) ligand 10 (CXCL10), chemokine (C–C motif) ligand 2 (CCL2), and chemokine (C–C motif) ligand 5 (CCL5) in infected INS‐1 cells. CVB2, 4, 5, and 6 lysed and replicated in INS‐1 cells; TCID₅₀ was lowest for CVB5 and highest for CVB6. IFN‐γ, CXCL10, and CCL5 mRNA levels all increased significantly following infection with CVB2, 4, 5, and 6 (P < 0.05). CCL2 mRNA increased with CVB2, 5, and 6 (P < 0.05), IFN‐α mRNA increased with CVB5 infection (P < 0.05), while TNF‐α mRNA and IFN‐β mRNA (P < 0.001) increased with CVB2 infection. Dose‐dependent effects of infection on cytokine mRNA levels were observed for all (P < 0.01) except IFN‐γ. Following inoculation of INS‐1 cells with CVB1 and 3, viral RNA was not detected and cytokine/chemokine mRNA levels were unchanged. In conclusion, CVB2, 4, 5, and 6 induce dose‐dependent cytokine and chemokine mRNA production from INS‐1 cells suggesting that pro‐inflammatory cytokine and chemokine secretion by beta cells is a potential mechanism for EV‐induced beta cell damage in type 1 diabetes. J. Med. Virol. 82:1950–1957, 2010. © 2010 Wiley‐Liss, Inc.