Abstract: Objective To combine Twisted file and Guttaflow gutta-percha in root canal therapy, compare it with the traditional root canal treatment of K file + gutta-percha lateral compaction in vivo and in vitro. Methods The clinical experiment included 120 cases of mandibular premolars treated with root canal therapy, with 60 patients separately in control ( K file ) group and experimental ( TF ) group. Root canals in the control group were filled with paste Cortisomol +0.02 tapered gutta-percha by lateral condensation after root canal preparation with K file in 0.02　taper. Root canals in the experimental group were filled with Guttaflow+ large tapered gutta-percha after root canal preparation with TF. Record postoperative pain conditions in two groups of patients, and review tooth condition 3, 6, 12 months after operation. The vitro experiment included 52 isolated mandibular premolars, with 26 teeth separately in control ( K file ) group and experimental ( TF ) group. The root canal treatment steps were the same with the clinical experiment. Dye penetration method was used to compare the micro leakage of root apices. Results One day after operation,the pain condition of experimental group was better than that of control group; there was no significant difference between the two groups in long term treatment effects 3 months after operation,but 6, 12 months later, the treatment effect in experimental group was better than that of control group. Dye penetration method used in in vitro experiment revealed that apical sealing of the experimental group was better than that of the control group.Conclusion The root canal therapy method of Twisted file + Guttaflow is better than K file + gutta-percha by lateral condensation. It is worthy of clinical application.