Background <p>Myeloid cell leukemia-1 (MCL-1)—an anti-apoptotic protein of the B-cell lymphoma 2 family—is commonly overexpressed in human cancers, promoting tumorigenesis and chemoresistance. Upregulated MCL-1 in human hepatocellular carcinoma (HCC) has been demonstrated in numerous studies, and therapeutic agents targeting this protein have been assessed. However, its prognostic significance in canine HCC remains unclear. The objective of this study was to detect MCL-1 protein in canine normal liver tissue and compare its expression level with that in HCC tissue using western blotting. Immunohistochemistry (IHC) was used to quantify MCL-1 intensity levels in normal, non-neoplastic hepatic diseases, and HCC tissues, and the differences were assessed. Additionally, the relevance of MCL-1 immunostaining to various clinical and pathological parameters was evaluated.</p> Results <p>MCL-1 expression was markedly elevated in HCC tissues relative to normal liver tissues (<i>P</i> = 0.029). Additionally, all 10 normal liver tissues exhibited low IHC expression, which significantly increased as the malignancy progressed (<i>P</i> &lt; 0.001). In the HCC samples, high MCL-1 immunostaining was substantially correlated with metastatic status (<i>P</i> = 0.034) and tumor size (<i>P</i> = 0.046). Moreover, survival curve analysis revealed a significant relationship between upregulated MCL-1 and lower disease-free survival and overall survival rate (<i>P</i> = 0.006 and <i>P</i> = 0.031, respectively).</p> Conclusion <p>MCL-1 expression is increased in canine HCC, and its overexpression significantly correlates to worse clinical outcomes. Therefore, MCL-1 is considered to be a promising prognostic marker.</p>