Background Increased NK activation has been associated with resistance to HIV-1 infection in several cohorts of HIV-1 exposed, uninfected subjects. Inheritance of protective NK receptor alleles (KIR3DS1 and KIR3DL1high) has also been observed in a subset of HIV-1 exposed, uninfected subjects. However, the exact mechanism contributing to NK activation in HIV-1 exposed, uninfected intra-venous drug users (EU-IDU) remains to be elucidated. Objective We investigated the role of both host genotype and pathogen-induced dendritic cell modulation of NK activation during high-risk activity in a cohort of 15 EU-IDU subjects and 15 control, uninfected donors from Philadelphia. Design We assessed the activation status of NK cells and Dendritic cells by flow cytometry and utilized functional assays of NK-DC cross-talk to characterize the innate immune compartment in EU-IDU subjects. Results As previously reported, NK cell activation (CD69) and/or degranulation (CD107a) was significantly increased in EU-IDU subjects compared to control uninfected donors (p=0.0056, n=13). Genotypic analysis indicated that the frequency of protective KIR (KIR3DS1) and HLA-Bw4*80I ligands was not enriched in our cohort of EU-IDU subjects. Rather, plasmacytoid dendritic cells (PDC) from EU-IDU exhibited heightened maturation (CD83) compared to control uninfected donors (p=0.0011, n=12). When stimulated in vitro, both PDCs and NK cells from EU-IDU subjects maintained strong effector cell function and did not exhibit signs of exhaustion. Conclusion Increased maturation of PDCs is associated with heightened NK activation in EU-IDU subjects suggesting that both members of the innate compartment may contribute to resistance from HIV-1 infection in EU-IDU.