Calcium Hydroxide (CH) is commonly employed as intracanal medicament in endodontics. In order to maximize its therapeutic effects, it is essential to develop new approaches for preparing the controlled drug release systems which, in turn, facilities the dissociation of CH into calcium and hydroxyl ions. This work studies the sustained-controlled release of calcium ions and the effect of pH changes on the different formulation of CH with hydrophilic natural polymers over a period of 30 days. Various formulations were prepared by combining CH with gelatin, aloe vera and gum tragacanth. Root canals of 60 human teeth were instrumented and filled with a different formulation of CH and suspended in plastic tubes containing distilled water. Three formulas of polymer/CH were evaluated, and pure CH powder was used as a control. At specific time intervals, the calcium ions release and the pH changes of the medium in different formulations were analyzed. The main interactions between the studied polymers and CH were investigated using FTIR spectra. The antibacterial activity of formulations against Enterococcus faecalis was also studied. Faster release of CH was observed for aloe vera/CH. Gum tragacanth/CH showed a slow-release during the first 15 days. In contrast, only Gelatin/CH formulation showed a prolonged release with statistically significant differences (P < 0.05). The pure CH showed significantly higher pH values than the other formulations. The Gelatin/CH formulation was a better sustained-release system than the pure CH, and it can be used as a promising vehicle for CH in the root canal treatment.