Objective: To investigate the effects of Er:YAG laser on the attachment of human periodontal ligament fibroblasts (hPDLFs) to denuded root surfaces simulating delayed replantation cases. Background data: Dental avulsion is one of the most severe dental traumas, which is often treated with replantation. In delayed replantation scenarios, poor prognosis, including root resorption, usually occurs due to poor root surface conditioning and nonviable hPDLF attachment. Methods: Thirty six root fragments (5 x 5 x 2 mm) were obtained from periodontium tissue-free premolar root surfaces. Specimens were randomly and equally assigned to the following: Group A, untreated control; Group B, 25 J/cm(2) and 10 Hz of Er:YAG laser irradiation; and Group C, 50 J/cm(2) and 10 Hz of Er:YAG laser irradiation. Some specimens in each group were then prepared for surface topography visualization under SEM, others were subjected to coculture with hPDLF suspension, and cell adhesion was further evaluated by SEM. Results: Group A presented homogenous smooth root surface, with fewer and round-shaped cells attached; Group B and C exhibited rather rough and irregular morphologies, and spindle-shaped fibroblasts were firmly attached by numerous lamellipodia and extensions. After a 3-day coculture, the number of fibroblasts attached in Group A was significantly lower compared with the other two laser-treated groups (p = 0.008 < 0.05). No significant alterations were observed between the two laser groups (p = 0.135 > 0.05). Conclusions: Er:YAG laser-treated root surfaces are compatible for the attachment of PDLFs, which suggests that Er:YAG laser irradiation may be used as a promising strategy for root surface conditioning in delayed replantation cases.