C Chitosan is a natural biopolymer from deacetylation of chitin which can be found in the horn beetle Xylotrupes gideon. Chitosan can be applied in dentistry because it is non-toxic, biocompatibility and biodegradability. Modification of chitosan into nano particles will increase diffusion and penetration of chitosan. Recent studies report that the effect of nano chitosan can increase the production of Reactive Oxygen Species (ROS) in cancer cells such as cervix cancer (HeLa), hepatic cancer (SMMC-7721), and breast cancer (MCF-7). This study aims to determine the effect of nano chitosan on the production of ROS in cancer cells HSC-3. The examination used nano chitosan with different concentration which were divided into four groups, 200ppm, 400ppm, 600ppm, and a group without exposure of nano chitosan. The results showed significant differences (p<0.05) between HSC-3 cells with exposure of nano chitosan compared to control group. Measurement of ROS using Fluorescence Microscope Zeiss Observer Z1, and analyzed using imageJ software. The result of ROS production, exposure of nano chitosan with concentration of 200ppm, 400ppm, and 600ppm was able to increase the production of ROS of HSC-3 cells. Nano chitosan with concentration of 400ppm has the most effect on HSC-3 cells, followed by 200ppm and 600ppm.