Background: Recent data indicated that macrophages may mutually interact with cancer cells to promote tumor progression and chemoresistance, but the interaction in cholangiocarcinoma (CCA) is obscure. Methods: 10x Genomics single-cell sequencing technology was used to identified the role of macrophages in CCA. Then, we measured the expression and prognostic role of macrophage markers and aPKC(iota) in 70 human CCA tissues. Moreover, we constructed monocyte-derived macrophages (MDMs) generated from peripheral blood monocytes (PBMCs) and polarized them into M1/M2 macrophages. A co-culture assay of the human CCA cell lines (TFK-1, EGI-1) and differentiated PBMCs-macrophages was established, and functional studies in vitro and in vivo was performed to explore the interaction between cancer cells and M2 macrophages. Furthermore, we established the cationic liposome-mediated co-delivery of gemcitabine and aPKC(iota)-siRNA and detect the antitumor effects in CCA. Results: M2 macrophage showed tumor-promoting properties in CCA. High levels of aPKC(iota) expression and M2 macrophage infiltration were associated with metastasis and poor prognosis in CCA patients. Moreover, CCA patients with low M2 macrophages infiltration or low aPKC(iota) expression benefited from postoperative gemcitabine-based chemotherapy. Further studies showed that M2 macrophages-derived TGF beta 1 induced epithelial-mesenchymal transition (EMT) and gemcitabine resistance in CCA cells through aPKC(iota)-mediated NF-kappa B signaling pathway. Reciprocally, CCL5 was secreted more by CCA cells undergoing aPKC(iota)-induced EMT and consequently modulated macrophage recruitment and polarization. Furthermore, the cationic liposome-mediated co-delivery of GEM and aPKC(iota)-siRNA significantly inhibited macrophages infiltration and CCA progression. Conclusion: our study demonstrates the role of Macrophages-aPKC(iota)-CCL5 Feedback Loop in CCA, and proposes a novel therapeutic strategy of aPKC(iota)-siRNA and GEM co-delivered by liposomes for CCA.