Objective: Endoscopic transforaminal lumbar interbody fusion (Endo-TLIF) has gained increasing popularity among spine surgeons. However, with the use of fluoroscopy, intraoperative radiation exposure remains a major concern. Here, we aim to introduce Endo-TLIF assisted by O-arm-based navigation and compare the results between O-arm navigation and fluoroscopy groups.Methods: Sixty-four patients were retrospectively analyzed from May 2019 to September 2020; the nonnavigation group comprised 34 patients, and the navigation group comprised 30 patients. Data on radiation dose, blood loss, postoperative drains, surgery time, complications, and length of hospital stay (LOS) were collected. Clinical outcomes were evaluated from postoperative data such as fusion rate, Oswestry Disability Index (ODI), and visual analogue scale (VAS). Radiation dose and surgery time were selected as primary outcomes; the others were second outcomes.Results: All patients were followed up for at least 12 months. No significant differences were detected in intraoperative hemorrhage, postoperative drains, hospital LOS, or complications between the 2 groups. The radiation dose was significantly lower in the navigation group compared with the nonnavigation group. The time of cannula placement and pedicle screw fixation was significantly reduced in the navigation group. No significant differences were detected between the clinical outcomes in the 2 groups (VAS and ODI scores).Conclusion: The present study demonstrates that O-arm-assisted Endo-TLIF is efficient and safe. Compared with fluoroscopy, O-arm navigation could reduce the radiation exposure and surgical time in Endo-TLIF surgery, with similar clinical outcomes. However, the higher doses exposed to patients remains a negative effect of this technology.