Purpose: To study the effect of Chitosan final irrigating solution compared with EDTA using different irrigation techniques, on TGF-β1 release from root canal dentin, using enzyme-linked immunosorbent assay (ELISA), and to evaluate the effect of released TGF-β1 on Dental Pulp Stem Cells. Materials and Methods: Human DPSCs were isolated from impacted third molars. Forty eight human root segments were prepared to a standardized truncated cone-shaped canal with an open apex of 1 mm diameter, the volume of prepared segments were determined using CBCT.Samples were randomized into two experimental groups (n=24) regarding the final irrigating solution; Group I: 1.5% NaOCl followed by 17% EDTA, Group II: 1.5% NaOCl followed by 1% Chitosan, then subdivided into subgroups A and B (n = 12) according to the final irrigation technique: A: Needle Irrigation, B: XP-Endo Finisher agitation. The released TGF-β1 were quantified using ELISA at 4 hours, 1 day, and 3 days following irrigation. DPSCs response to1.5% NaOCl, 17% EDTA, 1% Chitosan, and different concentrations of released TGF-β1 was assessed using MTT assay. Results: No statistically significant difference in the quantity of TGF-β1 released by 17% EDTA and 1% Chitosan following needle irrigation or XP Endo Finisher agitation.1% Chitosan significantly increased DPSCs viability compared to17% EDTA (P < 0.05).TGF-β1 showed significant expansion of viable DPSCs in a positive correlation to its concentration (P <0.001). Conclusions: 1% Chitosan effectively released TGF-β1 from root canal dentin as17% EDTA. The released TGF-β1 promoted DPSCs proliferation at picogram levels in a concentration dependent manner.