Objective: To preliminarily investigate the clinical outcomes of secondary loop electrosurgical excision procedure (LEEP) combined with transcervical resection of endocervical tissue (i.e., second combined surgeries) in patients with positive endocervical margins following the initial LEEP for high-grade squamous intraepithelial lesion (HSIL) or adenocarcinoma in situ (AIS) of the cervix. Methods: Patients who underwent second combined surgeries due to positive endocervical margins after the initial LEEP for cervical HSIL or AIS at Obstetrics and Gynecology Hospital, Fudan University between August 2015 and September 2023 were included. Postoperative cytological examinations, high-risk human papillomavirus (HR-HPV) testing, colposcopic biopsy results, and cervical canal length were followed up to evaluate the clinical efficacy of second combined surgeries. Results: (1) General clinical data: a total of 67 patients were enrolled, including 34 with cervical HSIL (HSIL group) and 33 with AIS (AIS group). In the HSIL group before the time of initial LEEP, the mean age was (41.3±5.3) years, with all patients positive for HR-HPV preoperatively. Preoperative cytology results revealed ≤low-grade squamous intraepithelial lesion (LSIL) in 13 cases and ≥HSIL in 21 cases. The preoperative cervical canal length was (3.71±0.17) cm. Patients in the AIS group before their the first LEEP were at an average age of (39.1±8.7) years old, with preoperative HR-HPV positive. Among them, 16 cases showed preoperative cytological results of ≤LSIL, while 17 cases showed ≥HSIL. The preoperative cervical canal length was (3.64±0.21) cm. (2) Pathological findings and postoperative follow-up of the HSIL group following second combined surgeries:in the HSIL group, the residual rate of HSIL in the endocervical canal tissue (ECT) was 24% (8/34). Out of the 34 HSIL patients, 10 cases (29%, 10/34) remained with positive endocervical margins post-second combined surgeries. Among these 10 patients, 5 cases (5/10) had no lesion detected in ECT, while the remaining 5 cases (5/10) exhibited HSIL in their ECT. Conversely, 24 patients (71%, 24/34) had negative endocervical margins after second combined surgeries. Of these 24 patients, 3 cases (12%, 3/24) were found to have HSIL in ECT, and 21 cases (88%, 21/24) had no lesion in ECT. During follow-ups conducted at 6 and 12 months post-second combined surgeries, the clearance rates of HR-HPV were 91% (31/34) and 100% (34/34), respectively. Notably, among the 29 patients (85%, 29/34) who were followed up for a period of 2 years or longer, all cases maintained a consistently negative HR-HPV status, highlighting the effectiveness of second combined surgeries in achieving long-term HR-HPV clearance (100%, 29/29). (3) Pathological findings and postoperative follow-up of the AIS group following second combined surgeries: the residual rate of AIS in the ECT following second combined surgeries among AIS patients was 15% (5/33). Out of the 33 AIS patients, 11 cases (33%, 11/33) had positive endocervical margins post-operation, among whom AIS was detected in the ECT of 2 cases (2/11), while 1 case (1/11) was diagnosed with adenocarcinoma in the cervical canal tissue (subsequently underwent radical surgery and was excluded from this study). In contrast, 22 patients (67%, 22/33) had negative endocervical margins post-operation, with AIS found in the ECT of 2 cases (9%, 2/22) and no lesions detected in the remaining 20 cases (91%, 20/22). Follow-up evaluations conducted at 6 and 12 months postoperatively revealed HR-HPV clearance rates of 91% (29/32) and 97% (31/32), respectively. All 32 (100%, 32/32) AIS patients were followed up for a duration of ≥2 years post-second combined surgeries, during which HR-HPV remained consistently negative. (4) Complications and cervical length following second combined surgeries: neither the HSIL group nor the AIS group experienced significant complications such as hemorrhage, infection or cervical canal adhesion. At the 6-month follow-up, the cervical length of both HSIL and AIS patients exceeded 3 cm. By the 12-month follow-up, the cervical length had recovered to 96.5% and 97.5% when compared to the original length, respectively, for the HSIL and AIS groups. Conclusions: For patients with HSIL or AIS who exhibit positive endocervical margins following the initial LEEP procedure, undergoing second combined surgeries presents as an optimal choice. This surgical intervention guarantees thorough excision of the lesion, and subsequent colposcopic follow-up evaluations consistently demonstrate an absence of residual disease or recurrence. Moreover, it augments the rate of sustained HR-HPV negativity, thereby contributing to more favorable clinical outcomes.