Vitamin D regulation is associated with several human disorders and contributes to various cellular mechanisms. Calcitriol (commercially available as Rocaltrol), an active Vitamin D metabolite, is known as a neuro-protective and anti-cancer drug but most importantly helps maintaining the calcium homeostasis inside the human body. The effectiveness of calcitriol to perform as an effective therapeutic agent is counteracted by its calcemic effects. In order to obtain better therapeutic results, synthetic calcitriol analogs without these calcemic effects have been recently developed but they are not yet cost-effective and their production is time-consuming. In order to determine the best active form of calcitriol that could provide higher chemotherapeutic activity without these calcemic effects, calcitriol mode of action was studied using yeast as a model system. In order to achieve this, we analyzed the calcitriol effects on yeast cellular growth based on calcium intake levels. In this work, we also assessed yeast strains with gene deletions of selected calcium transporter genes to understand the calcitriol metabolism. For the aim of understanding hypercalcemic effects of calcitriol, we developed a hypothesis based on calcitriol interactions with oxygen. Interestingly, use of an anaerobic model validated the oxygen interactions with calcitriol that might possibly cause calcemic effects on patients. Anaerobically grown yeast treated with calcitriol showed significantly less intracellular calcium levels when imaged under indo-1 calcium binding fluorescence dye as compared to calcitriol treated yeast grown under aerobic conditions. Finally, we predict that calcitriol might control free radical generation within the yeast system based on experiments with AAPH and UV- irradiation.