Background: Cholangiocarcinoma (CCA) is a type of cancer that originates from the biliary system. It typically has a subtle onset and high degree of malignancy, often leading to a terminal stage diagnosis, which limits the possibility of surgical intervention. Recent advancements in mitochondrial transfer therapy have enabled more precise treatment of CCA. This study aimed to investigate the effects and potential mechanisms of normal mitochondrial transplantation on the proliferation and energy metabolism of cholangiocarcinoma cells. Methods: The human cholangiocarcinoma cell line HuCCT1 was randomly divided into 5 groups: control (Con) group, mitochondrial transplantation 1 h (Mito-1 h) group, mitochondrial transplantation 6 h (Mito-6 h) group, mitochondrial transplantation 12 h (Mito-12 h) group, and mitochondrial transplantation 24 h (Mito-24 h) group. HuCCT1 cells received 143B wild-type mitochondrial fusion cells (143BρW)-derived mitochondria for different periods. Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was assessed using immunofluorescence and flow cytometry. Glycolytic activity was measured using commercial kits. RNA-seq was employed to analyze the differentially expressed genes (DEGs) in HuCCT1 cells following mitochondrial transplantation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were employed for further interpretation of the data. Protein expression levels were evaluated using western blot. Results: Normal mitochondria were successfully extracted from 143Bρw. Mitochondrial transplantation inhibited proliferation and promoted apoptosis of HuCCT1 cells. Western blot analysis suggested that mitochondrial transplantation significantly enhanced the expression of the mitochondrial apoptosis pathway, including Cytochrome C (Cyto-C), apoptosis-inducing factor (AIF), phosphorylated p53 (p-p53) and cleaved-Caspase-9. Moreover, mitochondrial transplantation enhanced the glucose uptake of HuCCT1 cells. Meanwhile, the levels of lactate acid, adenosine triphosphate (ATP), and Nicotinamide adenine dinucleotide phosphate (NADPH) were decreased by mitochondrial transplantation. Finally, RNA-seq results show that mitochondrial transplantation resulted in 60 significantly differentially expressed genes in HuCCT1 cells. Bioinformatics analysis indicated that the pentose phosphate pathway, Notch signaling pathway, Voltage-gated potassium channel complex, and insulin signaling are potential pathways for mitochondrial transplantation to inhibit CCA progression. Conclusions: Mitochondrial transplantation has the potential to impede cholangiocarcinoma cell proliferation by modulating energy metabolism, thus presenting a promising therapeutic strategy for this malignancy.