Background: The antibacterial photodynamic therapy (aPDT) has been used in dentistry againstoral microorganisms because of its excellent biocide effect. However, for carious lesions appli-cations, there is little evidence that this therapy is safe for the pulp tissue.Objective: This study evaluates the effects of an aPDT protocol on human pulp cells in vitro.Methods: Pulp cells isolated from dental pulp were exposed to an aPDT protocol associat-ing methylene blue (MB) at concentrations of 0.0125, 0.025 and 0.050 mg/ml and red laserirradiation using a continuous-wave indium-gallium-aluminum-phosphide (InGaAlP) diode laser( _ = 660 nm, 40 mW, 2.4 J, 60 J/cm2for 1 min). Pre-irradiation time was 5 min for each MB con-centration. Cell viability was determined by MTT assay and activity of alkaline phosphatase wasassessed by BCIP-NBT assay. Type of aPDT-induced cell death was assessed by flow cytometry.Data was statistically compared (ANOVA followed by Tukey’ or Bonferroni’s post hoc tests).Results: aPDT was able to kill pulp cells in a dye concentration-dependent manner. The cel-lular viability was significantly reduced when used MB at 0.025 or 0.050 mg/ml concentrations(p < 0.0001). At these concentrations, aPDT-induced cell death occurred mostly by necrosis.Alkaline phosphatase activity was significantly reduced in all experimental groups (p < 0.001).Pulp cells showed suitable viability when MB at 0.0125 mg/ml was exposed to laser irradiation.