Human cytomegalovirus (HCMV) is most likely to damage the central nervous system (CNS) during early embryonic development; however, the early neurodevelopmental abnormalities caused by HCMV infection and the regulation of cytokines remain unclear. Therefore, we investigated neuronal factors in the serum and cerebrospinal fluid (CSF) of newborns infected with HCMV using protein microarray technology with a view to elucidating the changes in specific neuronal factors for use in the development of a reliable index for predicting CNS injury caused by HCMV infection. Serum and CSF were collected from four newborns with HCMV infection and CNS injury (HCMV‐infected group) and from four newborns without CNS infection (control group). A protein microarray containing 29 kinds of CNS‐related cytokines was used to identify differentially expressed neuronal factors in the serum and CSF of the HCMV‐infected and control groups. The levels of the differentially expressed proteins were verified further in 30 CSF samples from an HCMV‐infected group using enzyme‐linkedimmunosorbent assay (ELISA). Between newborns in the HCMV‐infected and control groups, the protein microarray analysis identified three differentially expressed neurotrophic factors in the CSF samples: Acrp30, MMP‐3, and interleukin‐1 alpha (IL‐1α). No differential cytokine expression was seen in the serum. ELISA showed significantly higher expression levels of Acrp30 and MMP‐3 in the CSF of the 30 newborns with HCMV infection and CNS injury than in those in the control group, whereas the expression of IL‐1α was significantly lower. Our results demonstrate that changes in the expression levels of Acrp30, MMP‐3, and IL‐1α in the CSF of newborns infected with HCMV may be related to the pathogenesis of CNS infection.