Introduction: The aim of this study was to examine the effects of murine cytomegalovirus (MCMV) infection on neural stem/progenitor cells (NSPCs) in vitro. Materials and Methods: NSPCs infected by MCMV at a multiplicity of infections (MOIs) of 5, 1, or 0.1 were cultured in a differentiation medium. Ratio of differentiated cells was detected by flow cytometry. Protein markers for NSPCs and differentiated cell lineages were investigated with immunofluorescent staining. MCMV infection was traced by MCMV early antigen. MicroRNA expression of proteins related to Wnt signal pathways was measured with real-time RT-PCR. Results: Non-infected control NSPCs proliferated to form neurospheres and differentiated into neurofilament 200-positive neurons or glial fibrillary acidic protein-positive astrocytes. However, MCMV-infected NSPCs differentiated more slowly with less nestin. Moreover, the ratio of astrocytes, neurons, and mRNA levels of Wnt-3, Wnt-7a, Ngn-2, c-Myc, and cyclin D1 in infected NSPCs was significantly lower than the control group. Early antigens were consistently detected in infected NSPCs. These changes positively correlated with multiplicity of infections for the virus infection. Discussion: MCMV infection significantly inhibits differentiation of NSPCs and inactivates Wnt signal pathways during cell differentiation. This inhibitory effect may be a primary cause of brain development abnormalities in congenital cytomegalovirus infection.