This randomized controlled clinical trial aimed to evaluate the effect of the casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF) and photobiomodulation (PBM) in the treatment of dentin hypersensitivity (DH), and the impact of this on the health-related quality of life (HRQL). Methods Eighty teeth with DH were randomized into four groups and received three treatment sessions: PLACEBO = placebo + LASER application mimicking; CPP-ACPF = CPP-ACPF + LASER application mimicking; PBM = placebo + LASER active application; CPP-ACPF +PBM = CPP-ACPF + LASER active application. Tactile (exploratory probe) and evaporative (triple syringe) stimuli were used to measure DH and were recorded with the aid of a visual analogue scale (VAS) after the 1st, 2nd and 3rd treatment sessions and one-month follow-up. The HRQL was recorded in the DH experience questionnaire (DHEQ). Results The intragroup comparison showed a significant reduction in DH (p < 0.05) with both stimuli after one-month follow-up. The intergroup comparison with the evaporative stimulus showed that CPP-ACPF+PBM significantly reduced DH when compared to the rest of treatments, after one-month follow-up. CPP-ACPF+PBM group statistically differed from the other treatment groups in the DHEQ evaluation after one-month follow-up. Conclusion After one-month follow-up, the association of CPP-ACPF with PBM was effective in the reduction of DH and promoted a positive impact on the HRQL of the participants of this study. © 2019 Guanipa Ortiz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Ver menos