Gene expression in eukaryotes requires the post-transcriptional cleavage of mRNA precursors into mature mRNAs. The cleavage and polyadenylation specificity factor (CPSF) is critical for this process and its 73 kDa subunit (CPSF-73) mediates cleavage coupled to polyadenylation and histone pre-mRNA processing. Using CPSF-73 over-expression and siRNA-mediated knockdown experiments, this study identifies CPSF-73 as an important regulatory protein that represses the basal transcriptional activity of the HIV-1 LTR promoter. Similar results were found with over-expression of the CPSF-73 homologue RC-68, but not with CPSF 100 kDa subunit (CPSF-100) and RC-74. Chromatin immunoprecipitation assays revealed the physical interaction of CPSF-73 with the HIV-1 LTR promoter. Further experiments revealed indirect CPSF-73 binding to the region between -275 to -110 within the 5' upstream region. Functional assays revealed the importance for the 5' upstream region (-454 to -110) of the LTR for CPSF-73-mediated transcription repression. We also show that HIV-1 Tat protein interacts with CPSF-73 and counteracts its repressive activity on the HIV-1 LTR promoter. Our results clearly show a novel function for CPSF-73 and add another candidate protein for explaining the molecular mechanisms underlying HIV-1 latency.