<p>Xylazine, an alpha-2 adrenergic agonist originally developed as a veterinary sedative, has increasingly been detected as an adulterant in illicit drug supplies, particularly in combination with opioids such as fentanyl. This emerging pattern of misuse poses significant health risks and imposes a growing social burden, underscoring the urgent need to develop a rapid and comprehensive research strategy to investigate the potential toxicological mechanisms of xylazine. The present study aimed to comprehensively assess the toxicity of xylazine on the testis and its potential molecular mechanism using network toxicology and molecular docking analyses. By utilizing ChEMBL, STITCH, GeneCards, OMIM, TTD, and Drugbank databases, we predicted 126 xylazine-associated targets related to testis injury. Through further screening with STRING and Cytoscape software, 18 core targets in the testicular injury network, including steroid receptor coactivator (SRC) and Heat shock protein 90 alpha family class A member 1 (HSP90AA1), were obtained. GO and KEGG pathway analyses conducted through the DAVID database suggested that the core targets of xylazine-induced testicular toxicity may be enriched mainly in neuroactive ligand receptor interaction pathway, cancer pathways, apoptosis signaling pathways, and calcium signaling pathways. Molecular docking via AutoDock confirmed the strong binding between xylazine and the core targets. Together, these findings suggest that xylazine may be involved in regulating cell proliferation, cell apoptosis, and inflammation, which could potentially lead to testis injury. This study provides a theoretical basis for understanding the molecular mechanism of xylazine-induced testicular toxicity. In addition, network toxicology combined with molecular docking has provided new directions for the elucidation of the toxicity and mechanism of action of novel drugs.</p>