Background: Injection drug use (IDU) leads to immune system dysfunction, thereby increasing the risk of opportunistic infection. There is a critical need to reveal the role of IDU in the immunopathogenesis of HIV infection. Methods: We performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) derived from healthy control (HC) individuals, HIV-infected patients with IDU (HIV-IDU) and without IDU (HIVnIDU). In addition, the Gene Set Enrichment Analysis (GSEA) was used to analyze the immunomodulatory effects of differential immune cells. Results: Seven types of cells were identified with specific expressions of maker genes. Specific subsets such as CD14+ monocytes, plasmacytoid dendritic cells (pDCs), plasma cells, and CD8+ T cells displayed a high degree of heterogeneity among HC, HIV-nIDU, and HIV-IDU. We identified signature genes for each subset in distinct groups, including CFP+ CD14+ monocytes, PTPRCAP+ pDCs, IGHD+ plasma cells, and IFITM1+ CD8+T cells from HIV-IDU, whereas these genes were not expressed in such cells from HIV-nIDU. Moreover, considerable heterogeneity in the function of these immune cells was observed across different groups, especially the elevated IFN-alpha/(3 signaling for CD14+ monocytes, histone H2A/2B and H3/4 pathway for pDCs, the creation of C4 and C2 activators for plasma cells, and drug metabolism cytochrome p450 for CD8+ T cells in HIV-IDU individuals. Conclusion: Our comprehensive analyses clarify the heterogeneous characteristics of the immune landscape between HIV-IDU and HIV-nIDU. These insights provide a deeper understanding of the IDU-mediated immunopathogenesis in HIV infection.