Objective: This study aims to investigate the short-term effects of intraoperative electroacupuncture (EA) on peripheral blood lymphocyte subsets and postoperative pain in patients undergoing laparoscopic cholecystectomy. Methods: A single-center, prospective, randomized, controlled study was conducted. Sixty-six patients who underwent laparoscopic cholecystectomy were randomly assigned to the EA group (Group A) and control group (Group C). Patients in Group A received acupoint EA stimulation for 45 min after anesthesia induction. The stimulation points were bilateral Hegu (LI 4), Neiguan (PC 6), Yinlingquan (SP 9), and Zusanli (ST 36). In Group C, acupuncture needles were attached to the same acupoints, but not inserted into the skin. The primary outcome was the change in the proportion of lymphocyte subsets [CD3+, CD4+, CD8+, CD4+/CD8+, CD19+, and natural killer (NK) cells] measured by flow cytometry before surgery (at T0, before anesthesia induction) and after surgery (at T1, 5 min postsurgery). Additionally, the visual analog scale (VAS) was used to evaluate pain levels at 4 h after surgery. Results: All 66 randomized patients were included in the analysis. In Group A, the percentages of CD3+, CD4+, CD4+/CD8+, and CD19+ cells increased postsurgery (T1) compared with presurgery (T0) (p < 0.05), while the percentage of NK+ cells decreased (p < 0.05). No significant change in CD8+ cells was observed (p > 0.05). In Group C, no significant changes were found in peripheral blood lymphocyte subsets before and after surgery (p > 0.05). There were no significant differences between Group A and Group C at either time point (p > 0.05). At 4 h postsurgery, Group A had a significantly lower resting pain score (mean = 3.2) compared to Group C (mean = 4.6) (p < 0.05). No serious adverse events occurred. Conclusion: Intraoperative electroacupuncture may influence short-term distribution of certain lymphocyte subsets and reduce postoperative pain, but further studies with rigorous controls and extended follow-up are required to confirm its immunomodulatory effects.