Background: The genetic events responsible for tumor aggressiveness in endometrioid endometrial cancer (EEC) remain poorly understood. The chromosome 16q22 tumor suppressor genes CTCF and ZFHX3 are both frequently mutated in EEC, but their respective roles in outcome have not been determined. Methods: Targeted deep sequencing of CTCF and ZFHX3 was performed for 542 EEC samples. Copy number loss (CNL) was determined using microsatellite typing of paired tumor and normal DNA and a novel Bayesian method based on variant allele frequencies of germline polymorphisms. All statistical tests were two-sided. Results: Mutation rates for CTCF and ZFHX3 were 25.3% and 20.4%, respectively, and there was a statistically significant excess of tumors with mutation in both genes (P = .003). CNL rates were 17.4% for CTCF and 17.2% for ZFHX3, and the majority of CNLs included both CTCF and ZFHX3. Mutations were more frequent in tumors with microsatellite instability, and CNLs were more common in microsatellite-stable tumors (P < .001). Patients with ZFHX3 mutation and/or CNL had higher-grade tumors (P = .001), were older (P < .001), and tended to have more frequent lymphovascular space invasion (P = .07). These patients had reduced recurrence-free and overall survival (RFS: hazard ratio [HR] = 2.35, 95% confidence interval [CI] = 1.38 to 3.99, P = .007; OS: HR = 1.51, 95% CI = 1.11 to 2.07, P = .04). Conclusions: Our data demonstrate there is strong selection for inactivation of both CTCF and ZFHX3 in EEC. Mutation occurs at high frequency in microsatellite-unstable tumors, whereas CNLs are common in microsatellite-stable cancers. Loss of these two tumor suppressors is a frequent event in endometrial tumorigenesis, and ZFHX3 defects are associated with poor outcome.