Hormonal 1,25-dihydroxyvitamin D (1,25D) and its analogues signal through the nuclear vitamin D receptor (VDR). While numerous preclinical studies have provided evidence for antiproliferative and antitumour activities of 1,25D (analogues), such compounds have failed in clinical trials because of acquired tumour resistance. We previously found that the E3 ligase FBW7 interacts with the VDR. FBW7 regulates turnover of cell cycle drivers and is a tumour suppressor that is mutated in multiple malignancies, including head and neck squamous cell carcinoma (HNSCC). 1,25D accelerates the proteasomal degradation of FBW7 target proteins in a VDR- and FBW7-dependent manner. Surprisingly. FBW7 is also required for 1,25D-dependent transactivation. Here, we investigated the effects of FBW7 ablation by CRISPR/Cas9 editing on gene expression in 1,25D-sensitive human SCC25 HNSCC cells. We find that FBW7 loss induces the expression of several genes implicated in tumour progression and represses expression of those inhibiting tumour growth. Fold induction of the vast majority of the top 50 genes induced or repressed by 1,25D is attenuated by FBW7 ablation, suppressing 1,25D-driven regulation of genes implicated in cell proliferation and immune responses. Collectively, these data reveal that FBW7 is a critical cofactor of 1,25D signaling and provide a mechanism of acquired tumour resistance.