The activating transcription factor 1 (AP-1) family of proteins consists of a large number of inducible factors that are implicated in many biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. Here, we investigated the role of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the JC virus (JCV) promoter in glial cells. DNA binding studies demonstrated the specific association of c-Jun with its DNA sequences corresponding to the AP-1 site within the JCV promoter. Functional analysis of the promoter showed that ectopic expression of c-Jun and c-Fos results in an additive activation of the JCV early and late promoters. Further functional assays indicated that the JCV AP-1 binding site is sufficient to confer responsiveness to both c-Jun/c-Fos- and UV-induced activation when transposed to a heterologous promoter. Analysis of c-Jun expression during the viral infection cycle by Western blotting revealed that c-Jun is posttranslationally modified by phosphorylation and its protein level is substantially increased at the late phases of infection cycle. Altogether, our findings indicate that AP-1 family members may play a role in the pathogenesis of JCV-induced disease in the human brain by modulating JCV gene transcription.