In this report we directly examined the relative roles of NFAT5 and NF-κB p50/p65 in HIV-1 replication under conditions of activation by MTb co-infection, when NF-κB levels in the nucleus are elevated. We found that specific disruption of the NFAT5 binding site(s) in R5-tropic subtype B or subtype C infectious molecular clones significantly reduced virus replication in PBMC or MDM co-infected with a clinical isolate of MTb. Thus, NF-κB binding to the viral LTR is not sufficient to compensate for the loss of NFAT5 binding to the LTR under conditions of MTb co-infection. Reciprocally, an intact NFAT5 site could not compensate for disruption of the two NF-κB sites. Thus, both factors are required for MTb-induced HIV-1 replication.