Monkeypox virus (MPXV) is an emerging public health threat, with increasing epidemiological and clinical evidence supporting sexual transmission, including detection of viral DNA in semen. However, the pathogenic mechanisms of MPXV within the male reproductive system remain poorly defined. We employed a rhesus macaque model to investigate MPXV-induced testicular alterations. Combining bulk RNA sequencing, histopathological analysis and immunofluorescence staining, we characterized testicular pathology, viral antigen distribution, and host responses during the early stage of infection. We found that MPXV did not induce marked inflammation or widespread cell death, and seminiferous tubule architecture remained largely intact during infection. Furthermore, within seminiferous tubules, Sertoli cells showed higher proportion of MPXV antigen co-localization than spermatogonia and spermatocytes, whereas in the interstitium, vascular endothelial cells and immune cells, including macrophages and T cells, showed prominent MPXV antigen detection, consistent with a potential contribution of hematogenous dissemination to testicular involvement. MPXV infection was associated with downregulation of the Sertoli cell marker SOX9 and alterations in tight junction-associated proteins, suggesting changes in Sertoli cell characteristics and BTB-associated molecular features. Transcriptomic analysis revealed changes in interferon-related antiviral pathways and complement-related gene expression, with C1q-positive signals detected in both interstitial and seminiferous tubular regions. In addition, changes in spermatogenesis-related gene expression were observed, which may reflect alterations in the spermatogenic microenvironment. Collectively, this study provides a quantitative characterization of early MPXV-testis interactions and cellular tropism across multiple testicular cell types in a non-human primate model, and provides insights into the pathological and immune responses associated with MPXV infection.