Axin1, a regulator of Wnt signaling, was previously identified as playing a negative role in the late phase of human immunodeficiency virus type 1 (HIV-1) replication in HeLa-derived J111 cells. In this report, we studied the molecular mechanism of how Axin1 regulates HIV-1 replication. HIV-1 transactivator, Tat-dependent viral reporter gene expression was enhanced in J111 cells transfected with small interfering RNA (siRNA) against Axin1. In addition, viral transcription was upregulated in J111 cells transfected with siRNA against Axin1. In contrast, HIV-1 gene expression was not enhanced by transfecting HeLa cells with siRNA against Axin1. The expression levels of T cell factor-4 (TCF4) and beta-catenin were higher in J111 than HeLa cells. In addition, siRNAs against TCF4 and beta-catenin inhibited the Axin1 siRNA-dependent enhancement of HIV-1 gene expression in J111 cells. These results suggest that Axin1 plays a negative role in HIV-1 transcription through the Wnt signaling pathway in J111 cells under normal cell culture conditions.