Background: We undertook a randomized controlled trial to ascertain if single-incision laparoscopic cholecystectomy (SILC) was more beneficial for reducing postoperative pain than traditional laparoscopic cholecystectomy (TLC). Moreover, the influencing factors of SILC were analyzed. Methods: A total of 552 patients with symptomatic gallstones or polyps were allocated randomly to undergo SILC (n = 138) or TLC (n = 414). Data on postoperative pain score, operative time, complications, procedure conversion, and hospital costs were collected. After a 6-month follow-up, all data were analyzed using the intention-to-treat principle. Results: Among SILC group, 4 (2.9%) cases required conversion to TLC. Mean operative time of SILC was significantly longer than that of TLC (58.97 +/- 21.56 vs. 43.38 +/- 19.02 min, P < 0.001). The two groups showed no significant differences in analgesic dose, duration of hospital stay, or cost. Median pain scores were similar between the two groups 7 days after surgery, but SILC-treated patients had a significantly lower median pain score 6 h after surgery (10-point scale: 3 [ 2, 4] vs. 4 [ 3, 5], P = 0.009). Importantly, subgroup analyses of operative time for SILC showed that a longer operative time was associated with greater prevalence of pain score >5 (>= 100 min: 5/7 patients vs. <40 min, 3/16 patients, P = 0.015). Conclusions: The primary benefit of SILC appears to be slightly less pain immediately after surgery. Surgeon training seems to be important because the shorter operative time for SILC may elicit less pain immediately after surgery.