Although vitamin D (VD) is chemoprotective and enhances 5-fluorouracil (5-FU) cytotoxicity against colorectal cancer (CRC), little is known about its potential calcium (Ca )-mediated anti-tumorigenic actions. Therefore, this study compared between VD and its non-calcaemic analogue, Paricalcitol (Pcal), ± 5-FU in relation to chemoprevention and Ca -mediated apoptosis in vivo and in vitro. Seventy male mice were distributed to: negative controls, positive controls (PC), VD, Pcal, 5-FU, VD + 5-FU and Pcal+5-FU groups. All groups, except negative, received two consecutive azoxymethane (AOM)-injections (10 mg/Kg/week) for CRC induction. VD (1000 IU/kg; three times/week) and Pcal (1.25 μg/kg; three times/week) injections started week-16 post-AOM and for 10 weeks. Three successive 5-FU cycles began at week-21 (50 mg/Kg/week). Similar protocols with VD , Pcal and/or 5-FU were applied in the HT29 colon cancer cells. The PC group had abundant malignant tumours, markedly elevated proliferation markers (survivin/CCND1) and declines in cyclin-dependent kinase-inhibitor-1A, pro-apoptotic molecules (p53/BAX/cytochrome_C/caspase-3), tissue Ca concentrations and Ca -dependent proteins (CaSR/CAM/CAMKIIA). All monotherapies equally reduced tumour numbers and proliferation markers whilst promoting the anti-tumorigenic molecules. VD and/or 5-FU, but not Pcal monotherapy, enhanced Ca levels and Ca -related molecules (CaSR/CAM/CAMKIIA/BAX/cytochrome_C) in vivo and in vitro. However, VD + 5-FU co-therapy showed the lowest tumour numbers, the highest cell numbers in sub-G1 phase of cell cycle, alongside the most effective modulations of oncogenes, tumour suppressors and Ca -related molecules at the gene and protein levels in vivo and in vitro. VD was superior than Paricalcitol in potentiating 5-FU cytotoxicity, possibly by upregulating several Ca -related molecules involved in tumour suppression.