The Wnt signaling pathway plays an important role in neural cell development and function. The key components of this pathway, β-catenin and its partner TCF-4/LEF-1, exert their effects on transcription by entering the nuclei, where they associate with the TCF-4/LEF-1 DNA motif positioned in the promoters of several important genes. Here we examined the role of TCF-4 upon transcription of the human immunodeficiency virus type 1 (HIV-1) promoter in human astrocytic cells. Our results showed that expression of TCF-4 in human astrocytic cells (U-87MG cells) decreased the basal and Tat-mediated transcription of the HIV-1 long terminal repeat (LTR). Results from promoter deletion studies revealed that the promoter sequence of the LTR with no classical binding motif for TCF-4/LEF-1, which spans positions −80 to +80 of the LTR, remained responsive to down-regulation by TCF-4. Noticeably, removal of the sequences between positions −80 and −68 decreased the negative effect of TCF-4 on viral gene transcription. A mutant variant of TCF-4 with no binding site for β-catenin was able to down-regulate LTR transcription, suggesting that β-catenin may not be directly involved in the observed regulatory events. Results from the glutathione S-transferase pull-down assay as well as the combined immunoprecipitation and Western blot analysis of protein extract from U-87MG cells revealed an interaction of Tat with TCF-4. Subcellular examination of TCF-4 and Tat in cells expressing either protein alone showed a predominantly nuclear accumulation of these proteins. However, in cells which coexpressed both TCF-4 and Tat, significant levels of these proteins were found in the cytoplasm. All together, these observations provide evidence for the cooperative interaction of TCF-4, the important transcription factor of the Wnt pathway, with Tat; this interaction may determine the level of viral gene transcription in human astrocytic cells.