Root caries is an increasingly problem in aging societies with severe implications for the general health and wellbeing of large numbers of people. Strengthening type-I collagen, a major organic component of human dentin, has proved effective in preventing root caries. This study sought to determine whether exposure to riboflavin followed by UVA irradiation (RF/UVA) could promote additional collagen crosslinking, and thus improve the acid and enzymatic resistance of human dentin under simulated oral environments. If so, it could offer potential for treatment of the intractable problem of root caries. The greatest flexural strengths were found in dentin exposed to a 0.1% riboflavin solution for 1 minute followed by 1,600 mW/cm2 UVA irradiation for 10 minutes. Mineral loss and lesion depth were significantly lower in the RF/UVA group than in the control group. The microstructures of dentinal tubules and collagen networks after RF/UVA treatment retained their original forms after acidic and enzymatic degradation. In conclusion, RF/UVA treatment may be a new method for preventing root caries with promising prospects for clinical application.