Cholangiocarcinoma (CCA) is a prevalent and highly lethal form of cancer globally. Although microRNAs (miRNAs) have been implicated in the advancement of CCA, their potential influence on 5-fluorouracil (5-Fu) resistance in CCA remains to be fully elucidated. Here, in this study, we investigated the impact of miR-22-3p on CCA resistance. Our investigation involved bioinformatics analysis, which revealed an association between miR-22-3p and the progression, diagnosis, and patient survival of CCA. Furthermore, we validated a notable downregulation of miR-22-3p expression in CCA cell lines. Elevated levels of miR-22-3p inhibit the activity and proliferation of 5-Fu-resistant CCA cell lines. In addition, we confirmed that phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a target gene of miR-22-3p, and its expression correlates with the survival of CCA patients. Reduced PTEN expression enhances apoptosis in 5-Fu-resistant CCA cells. Meanwhile, we verified the existence of the miR-22-3p/PTEN/phosphatidylinositol-3 kinase (PI3K)/Protein kinase B (AKT) regulatory networks in CCA, influencing the sensitivity of CCA cells to 5-Fu. In conclusion, our findings suggest that miR-22-3p acts as a tumor suppressor. Its overexpression inhibits the PTEN/PI3K/AKT axis, promoting cell apoptosis and enhancing CCA sensitivity to 5-Fu.