The aim of this study was to evaluate the effects of UVA-activated riboflavin (UVA-RF) on the mechanical properties of non-demineralized human dentin. Dentin specimens obtained from 20 teeth were randomly divided into the following four groups: group 1 (control): no treatment, group 2 (low UVA-RF): specimens were exposed to UVA-RF for 10 min, group 3 (medium UVA-RF): specimens were exposed to UVA-RF for 30 min, and group 4 (high UVA-RF): specimens were exposed to UVA-RF for 60 min. Three-point flexural test and Raman spectroscopic analyses were performed. The mean flexural strengths (MPa) were 129.96, 128.96, 144.21, and 147.54, and the mean elastic modulus (GPa) were 8.59, 8.38, 10.21, and 9.87 for groups 1 to 4, respectively. Raman spectra showed chemical modifications of dental collagen under medium and high UVA-RF treatment. We conclude that medium and high UVA-RF increases the strength of non-demineralized human dentin by collagen crosslinking.