Magnesium-based biomaterials might provide an innovative therapeutic potential to substantially enhance regeneration of dental tissues. In previous work, magnesium oxide (MgO) has been studied for its potential ability to enhance cell attachment, proliferation rate and dentin matrix protein expression of human dental pulp cells (HDPCs). However, to date, dentinogenic effect of magnesium chloride (MgCl2) on cell viability and expression of extracellular matrix proteins in HDPCs has not been investigated. This study was designed to compare the stimulatory effect of different concentrations of MgCl2 on dentinogenesis of HDPCs. HDPCs were cultured with 0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM concentrations of supplemental MgCl2, 0 mM as negative control group. Stimulatory effect of MgCl2 was assessed by evaluating cell viability, and expression of dentin matrix proteins: dentin sialoprotein (DSP), dentin matrix protein1 (DMP-1), dentin sialophosphoprotein (DSPP) and type I collagen (COL-I). Statistical analysis was carried by Multi-Way Analysis of Variance (ANOVA) with Wilks’ lambda test. Supplemental MgCl2 concentration groups between 0.5 mM-4 mM elicited a significantly higher expression of DSP and DMP-1, while 0.5 mM-2 mM supplemental MgCl2 concentrations showed highest stimulatory effect on cell viability and, expression of DSPP, and COL-I, compared to the negative control group at all-time points (P<0.0001). However, 8 mM MgCl2 group had an inhibitory effect on HDPCs with significant lower cell viability and expression levels of DSP, DMP-1, DSPP, and COL-I compared to the control (P<0.0001). In conclusion, optimal (0.5 mM-2 mM) supplemental MgCl2 concentration groups significantly upregulated odontogenic differentiation with enhanced expression of dentin matrix proteins. This is the first study to reveal the dentinogenic effect of MgCl2 on dentin matrix protein expression in HDPCs. Magnesium-containing biomaterials may serve as a potential material for pulp repair and dentin regeneration.