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INTRODUCTION Guided tissue regeneration has been considered a promising biological strategy to replace conventional endodontic therapies of teeth with incomplete root formation. Therefore, in the present study, a collagen/gelatin hydrogel either containing dosages of fibronenctin (FN), or not, were developed and assessed concerning their bioactive and chemotactic potential on human apical papilla cells (hAPCs). METHODS Hydrogels were prepared by varying the ratio of collagen and gelatin (Col/Gel; v/v), and used to establish the following groups: Collagen (positive control); Col/Gel 4:6; Col/Gel 6:4; Col/Gel 8:2. The viability, adhesion and spreading of cells seeded on the hydrogels were evaluated. Different concentrations of FN (0, 5 or 10 μg/mL) were incorporated into the best formulation of the collagen/gelatin hydrogel selected. Then, the hAPCs seeded on the biomaterials were assessed concerning the cell migration, viability, adhesion and spreading, and gene expression of ITGA5, ITGAV, COL1A1 and COL3A1. RESULTS Col/Gel 8:2 group exhibited better cell viability, adhesion and spreading in comparison with Control. Higher values of hAPCs migration, viability, adhesion, spreading and gene expression of pulp regeneration markers were found, the higher the concentration was of FN incorporated into the collagen/gelatin hydrogel. CONCLUSION Collagen/gelatin hydrogel with 10 μg/mL of FN had potent bioactive and chemotactic effects on cultured hAPCs.