Objectives: Pleural mesothelioma (PM) is a rare disease with dismal outcome. Systemic treatment options include chemotherapy and immunotherapy, but biomarkers for treatment personalization are missing. The only FDA-approved diagnostic biomarker is the soluble mesothelin-related protein (SMRP). Krebs von den Lungen-6 (KL-6) is a human mucin 1 (MUC1) glycoprotein, which has shown diagnostic and prognostic value as a biomarker in other malignancies. The present study investigated whether KL-6 can serve as a diagnostic and/or prognostic biomarker in PM. Materials and methods: Using a fully-automated chemiluminescence enzyme immunoassay (CLEIA) for KL-6 and SMRP, pleural effusion samples from 87 consecutive patients with PM and 25 patients with non-malignant pleural disorders were studied. In addition, KL-6 and SMRP levels were determined in corresponding patient sera, and in an independent validation cohort (n = 122). MUC1 mRNA and protein expression, and KL-6 levels in cell line supernatants were investigated in PM primary cell lines in vitro. Results: PM patients had significantly higher KL-6 levels in pleural effusion than non-malignant controls (AUC 0.78, p < 0.0001). Among PM patients, levels were highest in those with epithelioid or biphasic histologies. There was a strong positive correlation between pleural effusion levels of KL-6 and SMRP (p < 0.0001). KL-6 levels in sera similarly associated with diagnosis of PM, however, to a lesser extent (AUC 0.71, p = 0.008). PM patients with high pleural effusion KL-6 levels (≥303 IU/mL) had significantly better overall survival (OS) compared to those with low KL-6 levels (HR 0.51, p = 0.004). Congruently, high tumor cell MUC1 mRNA expression in primary cell lines associated with prolonged corresponding patient OS (HR 0.35, p = 0.004). These findings were confirmed in an independent validation cohort. Conclusion: This is the first study demonstrating KL-6 as a potential novel liquid-based diagnostic and prognostic biomarker in PM.