H9c2 cell is a cardiac myoblastic cell line derived from rat heart tissue. Group B coxsackievirus (CVB) is the major pathogen of viral myocarditis and dilated cardiomyopathy. In this study, infectivity of CVB3 in H9c2 cells was observed to evaluate the value of H9c2 cells in the experimental study of CVB-induced myocardial diseases. H9c2 cells and cardiac and skeletal myocytes of Balb/c mouse and Sprague Dawley (SD) rat were infected with two CVB3 variants integrated with enhanced green fluorescence (EGFP) or Renilla luciferase (RLuc). The expressions of EGFP and RLuc were measured by fluorescence microscopy, flow cytometry, and chemiluminescence assays, and the viral genomic RNA level was detected by reverse transcriptase-polimerase chain reaction (RT-PCR). The results showed that neither EGFP expression nor RLuc expression could be detected in H9c2 cells infected with EGFP-CVB3 or RLuc-CVB3. The viral RNA could not be detected by RT-PCR from day 9 post-infection. The cardiac and skeletal myocytes of Balb/c mouse and cardiac myocytes of SD rat could be infected by CVB3, but the skeletal myocytes of SD rat and H9c2 cells could not. Taken together, this study suggests that H9c2 cell is not permissive cell for CVB3, and is not suitable for CVB-induced myocardial disease study. The results also support that H9c2 cells exhibit properties of skeletal myocytes from the view of infection.