The association between vitamin D and thyroid cancer is unclear. It is unknown if CYP27A1 or CYP2R1 are present in normal thyroid or cancer cells and there is limited information regarding response to treatment with vitamin D. SV40 immortalized follicular cells (N-thy) and six thyroid cancer cell lines were treated with 10 µm vitamin D₃, 0.1 µm 1,25(OH)₂D₃ or vehicle × 24 h. CYP27A1, CYP2R1, CYP27B1 and CYP24A1 mRNA were measured using quantitative real-time-PCR before and after treatment. Cell proliferation was also evaluated in TPC1 and C643 cells after treatment with D₃, 25(OH)D₃ and 1,25(OH)₂D₃. Baseline CYP27A1 and CYP27B1 mRNA were present in all cells, CYP2R1 was higher and CYP24A1 mRNA was lower in cancer cell lines versus N-thy. TPC1 cells had increased CYP24A1 mRNA levels when treated with both D₃ (3.49, p < 0.001) and 1,25(OH)₂D₃ (5.05, p < 0.001). C643 cells showed increased CYP24A1 mRNA expression when treated with 1,25(OH)₂D₃ (5.36, p < 0.001). D₃, 25(OH)D₃ and 1,25(OH)₂D₃ all significantly decreased cell proliferation in TPC1 and C643 cells. Overall, both cancerous and N-thy cell lines express CYP27A1 and CYP2R1 in addition to CYP27B1, establishing the potential to metabolize D₃ to 1,25(OH)₂D₃. Additionally, vitamin D₃, 25(OH)D₃ and 1,25(OH)₂D₃ all had an antiproliferative effect on two thyroid cancer cell lines.