Objective To compare the efficacy of one-time root canal therapy (ORCT) and multiple root canal therapy (RCT) in the treatment of acute pulpitis (AP). Methods The data of 362 AP patients (from January 2020 to March 2024) in our hospital were reviewed and divided into 2 groups according to different surgical protocols. A total of 181 patients receiving ORCT were classified as the ORCT group and 181 patients receiving RCT were classified as the RCT group. Compare the surgical success rate of the two groups, as well as periodontal health index [gingival index (GI), plaque index (PLI), depth of periodontal exploration (PD)], visual analogue scale (VAS) score, mastic function (mastic efficiency, bite force), inflammatory mediators level [(HIF-1α) and leukocyte differentiation antigen 14 (CD14), Toll-like receptor 4 (TLR4), CX3C chemokine ligand 1 (CX3CL1)], complication rate before and after surgery. Results There was no significant difference in the success rate of operation between the two groups (P > 0.05). Three months after operation, GI, PLI and PD in ORCT group were lower than those in RCT group (P < 0.05). The VAS score of ORCT group was lower than that of RCT group at 3 and 7 days after operation (P < 0.05). The masticatory efficiency and biting force of ORCT group were higher than those of RCT group 3 months after operation (P < 0.05). The levels of HIF-1α, CD14, TLR4 and CX3CL1 in ORCT group at 7 days after operation were lower than those in RCT group (P < 0.05). The complication rate of ORCT group was 3.87% (7/181) lower than that of RCT group (13.26% (24/181) (P < 0.05). Conclusion The surgical success rate of ORCT and RCT in AP patients is similar, but ORCT is more helpful to reduce postoperative pain, improve masticatory function, improve periodontal health, reduce inflammatory mediators and the risk of complications.