As the numbers of dentate elderly are globally increasing, prevention of root caries is becoming of paramount importance for the dental profession. Objectives: Since the effect of Er:YAG laser coupled with fluoride treatment in reducing root caries remains unknown, this study was aimed to investigate the preventive effect of combined fluoride and Er:YAG laser treatment at different energy densities on human root caries. Methods: Fifteen non-carious pre-molars were randomly selected and cut into halves. Each tooth half was varnished except two window areas, horizontally aligned and both treated with a 2.0% NaF gel. All the left windows received Er:YAG laser irradiation with energy levels of 5.1 J/cm2 (L1) or 10.2 J/cm2 (L2). Serving as controls, all the right windows were only treated with NaF gel. All the tooth halves went through a 2-day pH-cycling before thin sections were obtained and characterized using a polarized-light microscope (Olympus, Japan). The average lesion depths (ld) were enumerated and the preventive effect of laser was defined as µld, the difference between ld of control windows and that of the corresponding lased windows. One-sample and paired t-tests were used to evaluate the laser effects. Results: The results revealed significant preventive effects for both L1 and L2 groups, (both p<0.001). A greater µld (p<0.001) was observed in the L2 group (69.75μm±8.2) than that of the L1 group (55.60 μm±6.1). Conclusions: The Er:YAG laser irradiation has an additional effect on preventing root caries over and above that of NaF treatment in particular at the energy level of 10.2 J/cm2. This project was supported by grants, R-222000013112 and D-E01RP1222106 from Academic Research Fund, Ministry of Education, Singapore.