Objective To investigate the value of high resolution microendoscopy (HRME) for assessment of mucosal healing in patients with ulcerative colitis (UC) during clinical remission. Methods A total of 30 UC patients were randomly selected to collect HRME images. Pathological results were used as the gold standard to establish the diagnostic criteria of HRME on evaluation of colonic mucosal status. And then a prospective study was performed on 67 patients, who underwent standard endoscopy to evaluate colonic mucosal status and obtain biopsy specimens. The specimens were collected for HRME imaging. The accuracy of standard endoscopy and HRME in assessing ulcerative colonic mucosal healing was compared. Results According to the gland morphology, gland arrangement, gland opening and inflammatory cell infiltration, the HRME criteria for assessing UC mucosal status were formulated and divided into 0-3 grades with 4 levels. The prospective study of 67 patients with 113 sites showed that the sensitivity, specificity, positive predictive value, and negative predictive value of standard endoscopy for assessing mucosal healing in UC patients were 4468% (21/47), 9091% (60/66), 7778% (21/27), and 6977% (60/86), respectively, and the corresponding indicators of HRME imaging was 8723% (41/47), 9545% (63/66), 9318% (41/44), and 9130% (63/69), respectively. Compared with pathological results, the Kappa value of the consistency test of standard endoscopy and HRME imaging were 0379 and 0835, respectively (both P<005). Conclusion HRME can achieve real-time virtual pathological imaging of colonic mucosa for UC patients, which is more accurate than standard endoscopy for assessing mucosal healing.