HIV reservoirs persist despite successful antiretroviral therapy (ART) and are a major obstacle to the eradication and cure of HIV. The mature monocyte subset, CD14+CD16+, contributes to viral reservoirs and HIV-associated comorbidities. Only a subset of monocytes harbors HIV (HIV+), while the rest remain uninfected, exposed cells (HIVexp). We developed an innovative single cell RNA sequencing (scRNAseq) pipeline that detects HIV and host transcripts simultaneously, enabling us to examine differences between HIV+ and HIVexp mature monocytes. Using this, we characterized uninfected, HIV+, and HIVexp primary human mature monocytes with and without ART. We showed that HIV+ mature monocytes do not form their own cluster separately from HIVexp but can be distinguished by significant differential gene expression. We found that ART decreased levels of unspliced HIV transcripts potentially by modulating host transcriptional regulators shown to decrease viral infection and replication. We also identified and characterized mature monocyte subpopulations differentially impacted by HIV and ART. We identified genes dysregulated by ART in HIVexp monocytes compared to their uninfected counterpart and, of interest, the junctional protein ALCAM, suggesting that ART impacts monocyte functions. Our data provide a novel method for simultaneous detection of HIV and host transcripts. We identify potential targets, such as those genes whose expression is increased in HIV+ mature monocytes compared to HIVexp, to block their entry into tissues, preventing establishment/replenishment of HIV reservoirs even with ART, thereby reducing and/or eliminating viral burden and HIV-associated comorbidities. Our data also highlight the heterogeneity of mature monocyte subsets and their potential contributions to HIV pathogenesis in the ART era.IMPORTANCE HIV enters tissues early after infection, leading to establishment and persistence of HIV reservoirs despite antiretroviral therapy (ART). Viral reservoirs are a major obstacle to the eradication and cure of HIV. CD14+CD16+ (mature) monocytes may contribute to establishment and reseeding of reservoirs. A subset of monocytes, consisting mainly of CD14+CD16+ cells, harbors HIV (HIV+), while the rest remain uninfected, exposed cells (HIVexp). It is important to identify cells harboring virus to eliminate reservoirs. Using an innovative single-cell RNA sequencing (scRNAseq) pipeline to detect HIV and host transcripts simultaneously, we characterized HIV+ and HIVexp primary human mature monocytes with and without ART. HIV+ mature monocytes are not a unique subpopulation but rather can be distinguished from HIVexp by differential gene expression. We characterized mature monocyte subpopulations differently impacted by HIV and ART, highlighting their potential contributions to HIV-associated comorbidities. Our data propose therapeutic targets to block HIV+ monocyte entry into tissues, preventing establishment and replenishment of reservoirs even with ART.Keywords: HIV; HIV-associated comorbidities; latency; reservoirs; transcriptome.