Background: Pain intensity has been reported to fluctuate throughout the day in various clinical situations. This study aimed to evaluate the relationship between postoperative pain and circadian rhythm after pediatric acute appendicitis surgery. Methods: Two hundred patients, aged 6–18 years, undergoing acute appendicitis surgery were included in this prospective observational study. The patients were divided into four groups according to the time they underwent surgery: the night group, 01:01–07:00; morning group, 07:01–13:00; afternoon group, 13:01–19:00; and evening group, 19:01–01:00. Intraoperative and postoperative vital signs, postoperative 24-h Wong–Baker Faces Pain Rating Scale (FACEs) scores, and the amount of analgesic required were recorded. Results: A total of 186 patients were analyzed in the study. There was no statistically significant difference in the demographic characteristics of the patient groups. Additionally, no differences were observed in intraoperative and postoperative vital signs among the four groups. However, patients in the night group had significantly higher FACEs values than those in the other groups at each time point (1st, 3rd, 6th, and 12th h) up to 12 h (P = 0.007, P = 0.023, P = 0.048, and P = 0.003, respectively). The amount of analgesic required in the night group was statistically higher than in the other groups until 12 h (P = 0.002, P < 0.001, P = 0.002, and P = 0.004, respectively). Conclusion A relationship was found between acute appendicitis operations performed at night (01:01 to 07:00) under general anesthesia and circadian rhythm in children. We believe that considering circadian time in the relief of postoperative pain would be beneficial.