Endoscopic hand suturing (EHS) was first developed to firmly close a mucosal defect following endoscopic submucosal dissection and has the potential for expanding its application. This study aimed to investigate the feasibility and safety of EHS in various clinical settings. In this single-center pilot study, 15 patients who had diseases for potential indication of EHS were prospectively recruited. Technical success, clinical success after the procedure, and severe EHS-related adverse events were evaluated. EHS was applied for defect closure after gastric subepithelial lesion removal under laparoscopic observation (n = 9), defect closure after rectal endoscopic full-thickness resection (EFTR) (n = 2), defect closure after thoracoscopy-assisted esophageal EFTR (n = 1), mucosal closure for gastric ulcer bleeding (n = 1), mucosal closure after peroral endoscopic myotomy (POEM) (n = 1), and postoperative anastomotic leak (n = 1). EHS was completed without severe adverse events, and the clinical courses were also favorable in 13 (87%) patients. The median suturing time was 61 min. In patients with POEM and anastomotic leak, EHS was discontinued because of the narrow lumen. In conclusion, EHS appears feasible and safe in situations that require tissue apposition, whereas a small working space appears to hamper EHS.