MUC16/CA125 is associated with cancer proliferation in several tumor entities. The data on MUC16 expression in cholangiocarcinoma (CCA) tissue are very limited. The aim of this study was to assess the MUC16 status and its impact on survival in CCA patients. All the patients with surgically resected CCA that were diagnosed between August 2005 and December 2021 at the University Hospital Frankfurt were retrospectively analyzed. A 7-Mucin biomarker panel was assessed by immunohistochemistry. For overall survival (OS), Kaplan−Meier curves and Cox-regression analyses were performed. Randomly selected intrahepatic cholangiocarcinoma (iCCA) were further processed for differential expression profiling. A total of 168 patients with CCA were classified as MUC16 (−) (66%, n = 111) and MUC16 (+) (34%, n = 57). Subgroup analyses revealed a median OS of 56.1 months (95% CI = 42.4−69.9 months) and 27.4 months (95% CI = 15.8−39.1 months) for MUC16 (−) and MUC16 (+), respectively (p < 0.001). In multivariate analysis, MUC16 (+) (HR = 1.6, 95% CI = 1−2.6, p = 0.032) was an independent risk factor for poor prognosis. Prominently deregulated pathways have been identified following MUC16 expression, overrepresented in cell cycle and immune system exhaustion processes. These findings suggest including MUC16 in clinical routine diagnostics as well as studying its molecular pathways to identify further mechanistic key players.