New vitamin D analogs are interesting candidates for anticancer treatment, including squamous cell carcinomas (SCCs), especially in combination with cytostatics. In order to evaluate the effect of combined application of cisplatin, imatinib, or docetaxel and new vitamin D analogs [PRI-1906: (24E)-24a-homo-(1S)-1,25-dihydroxyergocalciferol, and PRI-2191: (24R)-1,24-dihydroxyvitamin D3], against the cells of two human SCCs lines (SCC-25 and FaDu), cytotoxic activity and the effect on cell cycle and apoptosis were determined. The synergistic or additive antiproliferative effect was observed for all cytostatics used after treatment of FaDu cell line with calcitriol or its analogs. Antagonism caused by combination of calcitriol and docetaxel was shown only in the lowest dose. FaDu cells treated with cytostatics and vitamin D analogs cumulated in G0/G1 stage. A statistically significant decrease (2x) in the percentage of apoptotic cells was observed only in combination of imatinib and calcitriol or PRI-1906. On the other hand, when the SCC-25 cell line incubated with cisplatin and imatinib in combination with calcitriol or PRI-2191 (100 nM) was used, the quantitative method of Chou and Talalay indicated antagonism. In the lower doses of calcitriol or PRI-2191 combined with imatinib, the synergistic effect was observed, but in the case of combination with cisplatin or docetaxel only weak additivity was detected. Moreover, a significant decrease (2x) of the percentage of SCC-25 cells undergoing apoptosis induced by docetaxel, cisplatin, and imatinib was observed. The combination of all cytostatic drugs applied with PRI-1906 in all doses caused synergism or additivity. These results might indicate that PRI-1906 is more effective than calcitriol or PRI-2191 as a potential anticancer agent, when used in combination therapy with cytostatic agents. To our knowledge, this is the first observation of interaction with calcitriol or its analogs and imatinib.