Background/Purpose Laparoscopic liver mono‐segmentectomy (LLMS) may improve patient outcomes, but it is difficult and its accuracy and safety are unknown. We evaluated the accuracy of LLMS using Glissonian approach with indocyanine green fluorescence (ICG) negative staining. Methods Seventy‐four patients eligible for LLMS except for segment 1 were enrolled. Preoperative three‐dimensional CT‐based surgical simulation was used to determine estimated liver resection volume (ELRV), which was compared with modified actual liver resection volume (ALRV) obtained from actual liver resection mass. The LLMS accuracy was also evaluated based on operator's experience (attending surgeon AS or trainee surgeon TS). Results Estimated liver resection volumes significantly correlated with ALRVs (r = .82) in all cases. Moreover, TS‐conducted LLMS also showed acceptable difference between ELRV and ALRV compared with AS‐conducted LLMS. There were no intergroup differences in estimated blood loss, operation time, time of Pringle maneuver, postoperative complications, and length of postoperative hospitalization (P < .05). Moreover, R0 resection was comparable between the AS and TS groups. Conclusions Laparoscopic liver mono‐segmentectomy with Glissonian approach using ICG negative imaging ensured safe and accurate procedure owing to facilitated visualization of the resection line. Our approach was effective in avoiding postoperative liver dysfunction and securing radical resection. In addition, it might be helpful in TS education of LLMS. Highlight Funamizu and colleagues compared the outcomes of laparoscopic liver mono‐segmentectomy using the Glissonian approach with indocyanine green fluorescence negative staining when conducted by attending surgeons and by trainee surgeons. The outcomes were acceptable without significant differences between the operator experience groups, indicating that the procedure is feasible, safe and accurate.