RING finger protein 4 (RNF4) represents a subclass of ubiquitin ligases that target proteins modified by the small ubiquitin‐like modifier (SUMO) for ubiquitin‐mediated degradation. We disrupted the RNF4 gene in chicken DT40 cells and found that the resulting RNF4^−/− cells gradually lost proliferation capability. Strikingly, this compromised proliferation was associated with an unprecedented cellular effect: the gradual decrease in the number of intact chromosomes. In the 6 weeks after gene targeting, there was a 25% reduction in the DNA content of the RNF4^−/− cells. Regarding trisomic chromosome 2, 60% of the RNF4^−/− cells lost one homologue, suggesting that DNA loss was mediated by whole chromosome loss. To determine the cause of this chromosome loss, we examined cell‐cycle checkpoint pathways. RNF4^−/− cells showed a partial defect in the spindle assembly checkpoint, premature dissociation of sister chromatids, and a marked increase in the number of lagging chromosomes at anaphase. Thus, combined defects in SAC and sister chromatid cohesion may result in increased lagging chromosomes, leading to chromosome loss without accompanying chromosome gain in RNF4^−/− cells. We therefore propose that RNF4 plays a novel role in preventing the loss of intact chromosomes and ensures the maintenance of chromosome integrity.