Latency is the main obstacle towards an HIV cure, with cure strategies aiming to either elicit or prevent viral reactivation. While these strategies have shown promise, they have only succeeded in modulating latency in a fraction of the latent HIV reservoir, suggesting that the mechanisms controlling HIV latency are not completely understood, and that comprehensive latency modulation will require targeting of multiple latency maintenance pathways. We show here that the transcriptional co-activator and the central mediator of canonical Wnt signaling, β-catenin, inhibits HIV transcription in CD4+ T cells via TCF-4 LTR binding sites. Further, we show that inhibiting the β-catenin pathway reactivates HIV in a primary TCM cell model of HIV latency, primary cells from cART-controlled HIV donors, and in CD4+ latent cell lines. β-catenin inhibition or activation also enhanced or inhibited the activity of several classes of HIV latency reversing agents, respectively, in these models, with significant synergy of β-catenin and each LRA class tested. In sum, we identify β-catenin as a novel regulator of HIV latency in vitro and ex vivo, adding new therapeutic targets that may be combined for comprehensive HIV latency modulation in HIV cure efforts. Author summary: There is no cure for HIV despite over 30 years of research. An HIV cure will rely on understanding cellular pathways that regulate HIV latency, which can be exploited for cure strategies. We report here for the first time that transcriptional co-activator β-catenin regulates HIV latency in the primary target for HIV latency, the CD4+ T cell population. We also show that its manipulation can reactive or repress HIV alone or in combination with known modulators of HIV latency. Together, our findings classify β-catenin as a regulator of HIV latency with significant applications to cure strategies. [ABSTRACT FROM AUTHOR]