Human cytomegalovirus (HCMV) is an opportunistic pathogen that causes severe diseases in congenitally infected newborns and immunocompromised patients. Currently, no vaccine is available to prevent HCMV infection. Anti-viral drugs are limited by their side effects and drug resistance. In this study, by performing a medium-sized, anti-HCMV chemical screening, we identified SP600125, CC-401, and the c-Jun N-terminal kinase (JNK) inhibitor VIII, three structurally different small molecule JNK inhibitors that effectively inhibited HCMV replication in cultured human fibroblasts (HFs). SP600125 showed its potential by inhibiting the viral replication of a HCMV laboratory strain in HFs and a HCMV clinical strain in human retinal pigment epithelial cells. Knockdown of JNK expression by RNA interference significantly impaired HCMV replication, mimicking the effect of the chemical inhibitors on virus infection. Mechanistically, SP600125 affects a very early step of the viral life cycle. Viral binding, entry, and the delivery of viral DNA into the cells were not inhibited by the compound. Instead, it suppressed the transcription of the immediate-early viral genes IE1/2 and the accumulation of their gene products. IE1/2 are among the first genes expressed after viral entry, and they are the master regulators of late phase viral gene expression. Consistent with this notion, the expression of other viral genes was also reduced after SP600125 treatment. We propose that JNK inhibitors have the potential to become a new class of anti-HCMV drug candidates, and JNK is a feasible target for the development of anti-HCMV drugs. J. Med. Virol. 87:2135–2144, 2015. © 2015 Wiley Periodicals, Inc.