Protein ADP-ribosylation is a reversible posttranslational modification of uncertain significance in cancer. In this study, we evaluated the consequences for cancer susceptibility in the mouse of a genetic deletion of the enzyme responsible for removing mono-ADP–ribose moieties from arginines in cellular proteins. Specifically, we analyzed cancer susceptibility in animals lacking the ADP-ribosylarginine hydrolase (ARH1) that cleaves the ADP ribose–protein bond. ARH1^−/− cells or ARH1^−/− cells overexpressing an inactive mutant ARH1 protein (ARH1^−/−+dm) had higher proliferation rates than either wild-type ARH1^+/+ cells or ARH1^−/− cells engineered to express the wild-type ARH1 enzyme. More significantly, ARH1^−/− and ARH1^+/− mice spontaneously developed lymphomas, adenocarcinomas, and metastases more frequently than wild-type ARH1^+/+ mice. In ARH1^+/− mice, we documented in all arising tumors mutation of the remaining wild-type allele (or loss of heterozygosity), illustrating the strict correlation that existed between tumor formation and absence of ARH1 gene function. Our findings show that proper control of protein ADP-ribosylation levels affected by ARH1 is essential for cancer suppression. Cancer Res; 71(15); 5327–35. ©2011 AACR.