AIMS: The progression of colorectal cancer (CRC) is driven by a small subset of cancer stem-like cells (CSCs), and mitochondrial function is essential for maintaining their stemness. TSP50, a novel identified oncogene, has been found to promote cell proliferation in multiple cancer types. In this study, we detected the regulatory role of TSP50 in regulating CSC-like properties and mitochondrial mass in CRC. MATERIALS AND METHODS: First, TSP50 expression and clinical relevance were analyzed via clinical databases and immunohistochemical (IHC). Subsequently, bioinformatic analyses, CRC cell lines, tumorsphere cultures, and mouse xenograft models were utilized to evaluate the relationship between TSP50 and CSC-like properties as well as mitochondrial mass. Finally, immunofluorescence, immunoprecipitation, and Western blotting were performed to dissect the regulatory mechanisms of TSP50, followed by rescue experiments conducted both in vitro and in vivo. KEY FINDINGS: TSP50 was overexpressed in CRC tissues, correlating with poor drug response and shorter overall survival (OS). Meanwhile, TSP50 was shown to enhance CSC-like properties in both CRC cells and mouse xenograft models, while concurrently increasing mitochondrial mass and reducing ROS levels, these effects were partially reversed by inhibition of the PI3K/AKT pathway. Mechanistic investigations revealed that TSP50-induced activation of PI3K/AKT signaling is primarily mediated by the enhanced catalytic activity of PI3K p110α subunit. SIGNIFICANCE: Collectively, TSP50 drives CRC malignancy by promoting CSC-like properties and enhancing mitochondrial function through PI3K/AKT signaling. These findings identify TSP50 as a potential therapeutic target for eliminating CSC-like cells and improving clinical outcomes in CRC treatment.