Background The effects of ethylenediaminetetraacetic acid (EDTA) on regenerative endodontic procedures (REPs) are controversial, because, despite releasing growth factors from dentine, some studies show negative effects on cell behaviour. Objectives The aim of the study was to investigate the influence of the use of EDTA in REP on the growth factors’ release, cell behaviour and tissue regeneration. Methods A systematic search was conducted (PubMed/Medline, Scopus, Cochrane Library, Web of Science, Embase, OpenGrey and reference lists) up to February 2021. Only in vivo and in vitro studies evaluating the effects of EDTA on the biological factors of dentine, pulp/periapical tissues and cell behaviour were eligible. Studies without a control group or available full text were excluded. The growth factors’ release was the primary outcome. Risk of bias in the in vitro and in vivo studies was performed according to Joanna Briggs Institute's Checklist and SYRCLE’s RoB tool, respectively. Results Of the 1848 articles retrieved, 36 were selected. Amongst these, 32 were in vitro, three animal studies and one with both models. The EDTA concentrations ranged from 3% to 15%, at different times. Regarding growth factors’ release (17 studies), 15 studies found significant transforming growth factor (TGF)‐β release after dentine conditioning with EDTA, and most found no influence on vascular endothelial growth factor release. Regarding cell behaviour (26 studies), eight studies showed no influence of EDTA‐treated dentine on cell viability, whereas, five, nine and six studies showed higher cell migration, adhesion and differentiation respectively. No influence of EDTA conditioning was observed in animal studies. In vitro studies had a low risk of bias, whereas animal studies had high risk of bias. Meta‐analysis was unfeasible. Discussion This review found that EDTA increased TGF‐β release and improved cell activity. However, well‐designed histological analyses using immature teeth models are needed. Conclusions High‐quality in vitro evidence suggests that EDTA‐treated dentine positively influences TGF‐β release, cell migration, attachment and differentiation; further research to evaluate its influence on tissue regeneration is necessary due to low methodological quality of the animal studies.