DNA-dependent protein kinase (DNA-PK), a nuclear protein kinase that requires association with DNA for its kinase activity, plays important roles in the regulation of different DNA transactions, mainly transcription, replication and DNA repair. We reported DNA-PK facilitated HIV transcription by interacting with and phosphorylating the carboxyl terminal domain (CTD) of the RNA polymerase II (RNAP II) complex recruited to HIV LTR. In our current study, we found that besides catalyzing directly CTD phosphorylation, DNA-PK mediates the recruitment of P-TEFb at HIV LTR. Accordingly, DNA-PK inhibition via highly specific small molecule inhibitors resulted in severe impairment of the phosphorylation of the serine 2 and serine 5 of the RNAP II CTD. Chromatin immunoprecipitation (ChIP) analysis showed that DNA-PK inhibition led to the establishment of transcriptionally repressive heterochromatin structures at the HIV LTR. Consequently, we found strong restriction to HIV transcription and replication by DNA-PK inhibitors. Similarly results were also obtained upon DNA-PK knockdown, validating the direct effect of inhibitors on DNA-PK. Moreover, we found that DNA-PK inhibitors successfully limit the reactivation of latent HIV proviruses in patients' PBMCs. This observation presents a strong evidence for the inclusion of transcription inhibitors, such as DNA-PK inhibitors as supplements to HAART regimens, in order to further enhance the restriction of HIV replication, besides limiting transcription from proviruses and resultant deleterious effects from viral proteins, such as in CNS. Thus, our results can be exploited for HIV “Cure” Research with Emphasis on Viral Suppression.