Background: Mid-regional pro-atrial natriuretic peptide (MR-proANP) is a strong prognostic biomarker in cardiovascular disease but there is limited data for its use among patients undergoing dialysis. Methods: This was a cohort study of patients receiving maintenance hemodialysis from two Danish centers. Blood sampling and echocardiography were performed before and after a dialysis session. We calculated the area under the curve (AUC) for the receiver operating characteristics for diagnosing heart failure and Cox regressions for cardiovascular events and all-cause mortality. Results: Of the 306 patients, 284 (93%) had MR-proANP measurements both before and after dialysis. Median concentration was 642 pmol/L (IQR 419–858) before and 351 pmol/L (IQR 197–537) after dialysis, a mean decrease of 330 pmol/L (43%, CI 296–364, P < 0.001). MR-proANP concentration both before and after dialysis was negatively correlated to left ventricular ejection fraction with no difference in predictive ability for heart failure, AUC before and after dialysis were 0.60 (CI 0.50–0.70) and 0.61 (CI 0.51–0.71) (P = 0.40). Median follow-up was 32 months (IQR 31–33), during which 99 patients (32%) had a cardiovascular event and 110 (36%) died. A doubling of MR-proANP concentration was associated with a hazard ratio (HR) of 1.6 (CI 1.3–1.9) before and 1.7 (CI 1.4–2.0) after dialysis for mortality and a HR of 1.5 (CI 1.2–1.9) before and 1.4 (CI 1.2–1.7) after dialysis for cardiovascular events (all P < 0.001). Conclusion: The MR-proANP concentration is elevated among patients undergoing hemodialysis and decreases during dialysis. MR-proANP concentration both before, after and intra-dialysis change strongly predicted cardiovascular events and all-cause mortality.