In this study, for the first time, transcriptomic analysis of canine Leydig cell tumors revealed 1500 differentially expressed genes, including 982 upregulated and 168 downregulated transcripts. Transcriptomic profiling was performed using RNA sequencing of formalin-fixed paraffin-embedded testicular tissues of healthy and Leydig cell tumor canine testes. Following differential expression analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were applied to identify dysregulated biological processes and signaling pathways. The analysis highlights key molecular pathways that drive tumor growth, specifically those controlling sex steroid production, blood vessel formation (angiogenesis), tumor microenvironment remodeling, and cholesterol metabolism. Crucially, the findings show significant similarities to human Leydig cell tumor mechanisms (the activation of the PI3K-Akt cell signaling pathway) while simultaneously identifying unique, previously understudied alterations in estrogen and relaxin signaling specific to dogs. Furthermore, the downregulation of genes responsible for cell differentiation and immune defense underscores how these tumors avoid normal cellular controls. In conclusion, these discoveries provide, for the first time, foundational molecular data that can improve canine oncological early diagnostics (e.g., biomarkers and targeted therapies), while establishing canine tissues as a valuable comparative model for studying testicular cancers in humans.