Efficient transcription of the HIV-1 genome is regulated by Tat, which recruits P-TEFb from the 7SK small nuclear ribonucleoprotein (snRNP) and other nucleoplasmic complexes to phosphorylate RNA polymerase II and other factors associated with the transcription complex. Although Tat activity is dependent on its binding to the viral TAR sequence, little is known about the cellular factors that might also assemble onto this region of the viral transcript. Here, we report that the splicing factor SRSF1 (SF2/ASF) and Tat recognize overlapping sequences within TAR and the 7SK RNA. SRSF1 expression can inhibit Tat transactivation by directly competing for its binding to TAR. Additionally, we provide evidence that SRSF1 can increase the basal level of viral transcription in the absence of Tat. We propose that SRSF1 activates transcription in the early stages of viral infection by recruiting P-TEFb to TAR from the 7SK snRNP. Whereas in the later stages, Tat substitutes for SRSF1 by promoting release of the stalled polymerase and more efficient transcriptional elongation.