In the case of complete luxation with dental trauma, delayed replantation leads to a higher probability of root resorption. We evaluated the efficacy of N-acetylcysteine (NAC) as a root canal medication for trauma-associated root resorption. A rat dental trauma model was used. In the test group, mesial root pulpectomy was carried out after tooth extraction. NAC was applied as a root canal medication followed by replantation. We euthanized the rats after two weeks, excised the maxilla, and prepared thin sections, followed by H-E and TRAP staining. Furthermore, RAW264.7 cells were treated with RANKL and NAC. The effects of NAC on osteoclast differentiation were investigated by determining the numbers of osteoclasts and nuclei and quantifying osteoclast-related mRNA expression levels by real-time PCR. The level of root resorption and number of odontoclasts tended to be lower in the NAC groups than in the control group. In terms of osteoclast differentiation, the total number of osteoclasts was lower in both the 5 and 10 mmol/L NAC groups than in the control group. Real-time PCR revealed that the expression levels of RANK , NFATc1 , DC-STAMP , CD47 , and cathepsin K were lower in the 10 mmol/L NAC group. NAC tended to inhibit root resorption and reduce the number of odontoclasts. NAC also inhibited the expression of osteoclast-related genes. Overall, NAC reduced the number of odontoclasts, inhibited bone resorption, and influenced the inhibition of osteoclast differentiation, fusion, and functions. Therefore, we suggest that NAC may be beneficial in the treatment of root resorption.