Cholangiocarcinoma (CCA) is a lethal malignancy in the hepatobiliary system, with dysregulated protein expression and phosphorylation signaling. However, the protein and phosphorylation signatures of CCAs are little-known. Here, we performed the proteomic and phosphoproteomic profiling of tumors and normal adjacent tissues (NATs) from patients with CCA and predicted eleven PKs high-potentially related to CCA with a comprehensive inference of the functional protein kinases (PKs) (CifPK) pipeline. Besides the two known CCA-associated PKs, we screened the remaining candidates and uncovered five PKs as novel regulators in CCA. Specifically, the protein kinase D (PKD) family members, including PRKD1, PRKD2, and PRKD3, were identified as critical regulators in CCA. Moreover, the pan-inhibitor of the PKD family, 1-naphthyl PP1 (1-NA-PP1), was validated as a potent agent for inhibiting the proliferation, migration, and invasion ability of CCA cells. This study reveals new PKs associated with CCA and suggests PRKD kinases as novel treatment targets for CCA.