Identification of cellular proteins, in addition to already known transcription factors such as NF‐κB, Sp1, C‐EBPβ, NFAT, ATF/CREB, and LEF‐1, which interact with the HIV‐1 LTR, is critical in understanding the mechanism of HIV‐1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV‐1SF162 infection of monocyte/macrophages and reactivation of HIV‐1 in U1 cells, a macrophage model of latency. We observed that HIV‐1SF162 infection of monocyte/macrophages and reactivation of HIV‐1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA‐induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA‐induced U1 cells in comparison to PMA‐induced U937 cells. We focused on understanding the potential role of PKM2 in HIV‐1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA‐activated U1 and TZM‐bl cells demonstrated the interaction of PKM2 with the HIV‐1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV‐1 LTR‐luciferase reporter in U937, U‐87 MG, and TZM‐bl cells. Using various truncated constructs of the HIV‐1 LTR, we mapped the region spanning −120 bp to −80 bp to be essential for PKM2‐mediated transactivation. This region contains the NF‐κB binding site and deletion of this site attenuated PKM2‐mediated activation of HIV‐1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF‐κB. These observations demonstrate for the first time that PKM2 is a transcriptional co‐activator of HIV‐1 LTR. J. Cell. Physiol. 232: 517–525, 2017. © 2016 Wiley Periodicals, Inc. PKM2 is a transcriptional co‐activator of HIV‐1 LTR.