The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin–proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/β-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole–based TNKS1/2 inhibitor OM-153 reduces WNT/β-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33–10 mg/kg twice daily. In addition, OM-153 potentiates anti–programmed cell death protein 1 (anti–PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral–twice daily administration of 100 mg/kg. In contrast, mice treated oral–twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153–mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations. Significance: This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models.