Cyclin D1 is frequently overexpressed in esophageal squamous cell carcinoma (ESCC) and is considered a key driver of this disease. Mutations in FBXO4, F-box specificity factor that directs SCF-mediated ubiquitylation of cyclin D1, occur in ESCC with concurrent overexpression of cyclin D1 suggesting a potential tumor suppressor role for FBXO4. To evaluate the contribution of FBXO4-dependent regulation cyclin D1 in esophageal squamous cell homeostasis, we exposed FBXO4 knockout mice to N-nitrosomethylbenzylamine (NMBA), an esophageal carcinogen. Our results revealed that loss of FBXO4 function facilitates NMBA induced papillomas in FBXO4 het (+/-) and null (-/-) mice both by numbers and sizes 11 months after single dose NMBA treatment at 2mg/kg by gavage when compared to that in wt (+/+) mice (P < 0.01). No significant difference was noted between heterozygous or nullizygous mice consistent with previous work. To assess cyclin D1/CDK4 dependence, mice were treated with the CDK4/6 specific inhibitor, PD0332991, for 4 weeks. PD0332991 treatment (150mg/kg daily), reduced tumor size and tumor number. Collectively, our data support a role for FBXO4 as a suppressor of esophageal tumorigenesis.