ABSTRACT: Introduction: Repeated exposure to HIV-1 usually result in infection; however, some individuals, under constant exposure to the virus and against the statistical probability, do not show serological or clinical evidence of infection. These individuals are known as HESN (HIV-Exposed Seronegative) and have turned into a key population to search of the natural resistance phenomenon against HIV-1 infection. HESN studies include persons with high-risk sexual behaviors, such as Men Who Have Sex with Men (MSM), who experience biological and sociocultural issues that contribute to a higher risk of acquiring the HIV-1 infection. MSM has been poorly studied compared to other cohorts, such as commercial sex workers (CSW) or intravenous drug users (IDU). Two decades ago, evidence regarding the role of NK cells in natural resistance to HIV-1 infection such as higher effector capacity and higher cytokine production was described in HESN. However, little is known about the effector and antiviral potential of NK cells in the elimination of HIV-infected CD4+ T cells in high-risk MSM. For this, we propose to evaluate the role of NK cells in the natural resistance to HIV-1 infection in this high-risk population. Methodology: MSM at high-risk (HR-MSM) and low risk (LR-MSM) of HIV-1 infection were included in the study. The effector and antiviral capacity of NK cells were evaluated in co-cultures with HIV-infected CD4+ T cells. The p24 viral protein was quantified by intracellular cytometry in HIV-1-infected CD4+ T cells and the supernatants of the co-culture, by ELISA. Activation markers, cytokine production, and functional capacity of NK cells were evaluated by flow cytometry, and the quantification of cytokines in the co-culture was measured by Cytometric Bead Array (CBA). Results: MSM at high-risk of HIV-1 infection exhibit a higher capacity to eliminate HIV+ CD4+ T cells, a reduced percentage of CD69+, and a higher percentage of NKG2D+ NK cells. Despite the lower levels of CD69+ NK cells, within this population, higher percentages of CD69+ IFN-γ+ and CD69+ NKG2D+ NK cells were found in the HR-MSM group. In addition, higher levels of RANTES and Granzyme B were found in the supernatants of the co-cultures of HR-MSM; these molecules are linked to the antiviral activity, which could explain the lower percentages of p24+ cells and the reduced concentration found for this protein in the evaluated supernatants. Finally, correlations among a reduction in the percentage of p24+ cells, RANTES concentration, and percentage of positive NKG2D NK cells were found with the number of sexual partners in the last three months. Conclusion: NK cells from HR-MSM individuals had a higher antiviral capacity, resulting in a lower concentration of p24 protein in the co-cultures and the percentage of p24+ CD4+ T cells. Changes on the antiviral capacity could be driven by differences found in the activation markers and the chemokines and cytokines production compared to LR-MSM. The production of some antiviral molecules was also correlated with the number of sexual partners in the last three months. Altogether, this information suggests that higher exposure to HIV-1 in high-risk individuals could be included in a “training” state of NK cells to face the virus, influencing the natural resistance to HIV observed in this population.