Objective      To study the role of calretinin (CR) immunohistochemical and acetylcholinesterase (AchE) staining in pathological diagnosis of Hirschsprung disease (HD) and the role of CR immunohistochemical staining in pathological diagnosis of HD when the rectum mucosal biopsy samples were inadequate.       Methods      The accuracy and sensitivity of CR immunohistochemical staining of rectum mucosal biopsy samples from 163 HD patients were retrospectively studied and compared. The accuracy of CR immunohistochemical staining of rectum mucosal biopsy samples containing 1 submucosal nerve plexus was studied in pathological diagnosis of HD in 26 patients.       Results      The sensitivity of CR immunohistochemical staining was significantly higher than AchE staining for the diagnosis of HD in patients aged 3 months. The CR immunohistochemical staining of rectum mucosal biopsy samples containing 1 submucosal nerve plexus could accurately diagnose HD in 21 patients.       Conclusion      CR immunohistochemical staining of rectum mucosal biopsy samples is better than AchE staining of rectum mucosal biopsy samples in diagnosis of HD.