This study aimed to evaluate the in vivo biocompatibility and periodontal regenerative potential of enzymatically solidified chitosan hydrogels with or without incorporated periodontal ligament cells (PDLCs). To this end, chitosan hydrogels, with (n=8; CHIT+CELL) or without (n=8; CHIT) fluorescently-labeled PDLCs, were prepared and transplanted into rat intrabony periodontal defects; untreated defects were used as empty controls (n=8; EMPTY). After 4 weeks, maxillae were harvested, decalcified and used for histological, histomorphometrical and immunohistochemical assessments. The results showed that PDLCs remained viable upon encapsulation within chitosan hydrogels before transplantation. Histological analysis demonstrated that the chitosan hydrogels were largely degraded after 4 weeks of implantation, without any adverse reaction in the surrounding tissue. In terms of periodontal regeneration, alveolar bone height, alveolar bone area and epithelial downgrowth were comparable for CHIT, CHIT+CELL as well as EMPTY groups. In contrast, both CHIT and CHIT+CELL showed a significant increase in functional ligament length compared to EMPTY. From a cellular perspective, the contribution of chitosan hydrogel incorporated cells to the periodontal regeneration could not be ascertained, as no signal from transplanted PDLCs could be detected at 4 weeks post-transplantation. The results demonstrated that enzymatically solidified chitosan hydrogels are highly biocompatible and biodegradable. Moreover, chitosan hydrogels without cell loading can improve periodontal regeneration in terms of functional ligament length, indicating the great potential of this hydrogel in clinical applications. Further work on the use of chitosan hydrogels as cell carriers is required.