Background/Aim: The hormonally-active form of vitamin D, 1,25(OH)(2)D-3, demonstrated activity against oral squamous cell carcinoma (OSCC). Cytochrome P450scc (CYP11A1)-derived vitamin D hydroxyderivatives, such as 20(OH)D-3 and 1,20(OH)(2)D-3, have overlapping beneficial effects with 1,25(OH)(2)D-3 without causing hypercalcemia. This study sought to determine (i) whether 20(OH)D-3 and 1,20(OH)(2)D-3 exhibit antitumor effects against OSCC comparable to those of 1,25(OH)(2)D-3 and (ii) whether these effects may stem from down-regulation of sonic hedgehog (SHH) or WNT/beta-catenin signaling pathways. Materials and Methods: Effects on CAL-27 cells were assessed by 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium inner salt and spheroid assays. Signaling pathways were assessed by immunofluorescence and western blotting. Results: 20(OH)D-3 and 1,20(OH)(2)D-3 inhibited the growth of CAL-27 and demonstrated inhibition of WNT/beta-catenin and the SHH signaling as evidenced by down-regulation of nuclear translocation of glioma-associated oncogene 1(GLI1) and beta-catenin. Conclusion: Noncalcemic vitamin D hydroxyderivatives demonstrated antitumor activities against OSCC comparable to those of 1,25(OH)(2)D-3. Their activities against SHH and the WNT/beta-catenin pathways provide insight for a possible target for OSCC treatment.