Introduction:Propolis trigona contains minerals and flavonoids. Minerals and flavonoids affect the tooth enamel hardness. The presence of flavonoids in propolis which has been proven to be able to suppress the formation of bacterial biofilms that cause caries is expected to have an effect on hardness of tooth enamel so that caries can be prevented. This work was aimed to determine the effect of nano Propolis trigona sp (±77,3 nm) on enamel hardness of permanent molar human tooth in vitro. Methods: The sample used in this study were molars after extraction which were caries-free, without anomalies and had no fillings. The molars were sterilized using chlorhexidine gluconate (CHG) 0.1% and phosphate buffer saline (PBS). The twenty-four molar specimen were selected and divided into three treatment and a control group. The specimens were smeared with nano propolis 30%, 40%, 50% and the con-trol group was smeared with fluor protector, for 45 min in the solution. The hardness of tooth enamel was measured using the vickers micro-hardness Testing machine with force 300 grams for 10 seconds, prior and after treatment with the testing material. Results: The harden of enamel was clearly observed in a dose. The harden of enamel was most evidence in the treatment group suggesting a dose-dependent manner. Significant hardness of enamel was observed in the molar teeth treated with 30% and 50% nanopropolis compared to the control group. Conclusion: Thus, it can be concluded that nano propolis affect the improvement of molar enamel hardness, and there should be further studies on the optimal time usage of nano propolis on the tooth.