© 2016 American Medical Association. All rights reserved. IMPORTANCE Previous pharmacogenetic studies have shown the prognostic impact of several rare dihydropyrimidine dehydrogenase gene (DPYD) variants on fluorouracil-related adverse events (fluorouracil AEs). However, conflicting results highlight the need for prospective validation in large, homogeneous patient populations uniformly treated with current standard combination therapies used in colon cancer (CC). OBJECTIVE To determine the impact of DPYD variants on fluorouracil AEs in patients with stage III CC treated with a fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) regimen. DESIGN, SETTING, AND PARTICIPANTS Pharmacogenetic substudy of 1545 patients who participated from December 2005 to November 2009 in the European Pan-European Trials in Alimentary Tract Cancer (PETACC)-8 randomized phase 3 clinical trial. INTERVENTIONS Patients with resected stage III CC were randomized to receive standard adjuvant FOLFOX4 alone or FOLFOX4 combined with cetuximab for 6 months. MAIN OUTCOMES AND MEASURES Patientswere genotyped on 25 DPYD variants.We tested the individual associations between each DPYD variant and grade 3 or greater fluorouracil AEs. RESULTS A total of 1545 patients (57.6%male; median [range] age, 60 [19-75] years) were included in the analysis. The incidence of grade 3 or greater fluorouracil AEs in D949V and V732I (DPYD∗6) carriers was 18 in 21 (85.7%) and 121 in 199 (60.8%), respectively. After adjusting for multiple variables, statistically significant associations were identified between grade 3 or greater fluorouracil AEs and both D949V (odds ratio [OR], 6.3 [95%CI, 2.0-27.0]; P < .001) and V732I variants (OR, 1.7 [95%CI, 1.3-2.4]; P < .001). Grade 3 or greater overall hematologic adverse events were associated with V732I (OR, 1.9 [95%CI, 1.4-2.6]) and D949V (OR, 5.2 [95%CI, 2.0-16.0]), and V732I was associated with grade 3 or greater neutropenia (OR, 1.8 [95%CI, 1.3-2.4]). The association of V732I with the occurrence of grade 3 or greater fluorouracil AEs and overall hematologic adverse events was validated in an independent cohort of 339 patients with metastatic colorectal cancer receiving FOLFOX4 in the Fédération Francophone de Cancérologie Digestive 2000-05 phase 3 trial. CONCLUSIONS AND RELEVANCE In this large phase 3 study, statistically significant associations were found between DPYD variants (D949V and V732I) and increased incidence of grade 3 or greater fluorouracil AEs in patients treated with adjuvant fluorouracil-based combination chemotherapy. Further studies are warranted to confirm and quantitate these associations.