Abstract Objective: To evaluate the effects of HIV-1 infection on the general condition, viral replication, and tissue distribution of mpox virus (MPXV) in humanized mice, and to provide an experimental model and data for studying the pathogenesis of MPXV in the context of HIV-1 infection. Methods: Immunodeficient NPG mice reconstituted with human hematopoietic stem cells (Hu-HSC-NPG mice) were first inoculated with HIV-1JRCSF to establish stable infection. On day 35 post-HIV-1 infection, the mice were sequentially inoculated with MPXV. Body weight changes, survival rates, plasma viral loads, tissue HIV-1 DNA expression, and MPXV DNA distribution were systematically monitored in this experiment. Results: Following HIV-1JRCSF inoculation, mice body weight slowly declined to 95.09% of the initial weight by day 35 post-infection. and the proportion of CD4+ T cells also significantly decreased. After MPXV challenge, body weight showed mild fluctuations. No deaths occurred throughout the observation period. HIV-1 RNA peaked at day 14 post-infection (105.72 copies/mL) and then entered a plateau phase. MPXV DNA continuously increased to 106.12 copies/mL within 21 days post-MPXV infection. HIV-1 DNA was widely distributed in the heart, liver, spleen, lungs, kidneys, and brain. MPXV DNA loads in various tissues progressively increased over time. Conclusion: In HIV-1-infected humanized Hu-HSC-NPG mice, MPXV loads show a persistent increase in plasma and multiple organs, and HIV-1 DNA is detectable in all examined tissues. This model effectively recapitulates HIV-1-induced tissue viral reservoirs and systemic MPXV infection, providing reliable experimental data and an ideal small animal model for further investigation of MPXV pathogenesis in the setting of HIV-1 infection.