The aim of this study is to elucidate the differences of the clinicopathological characteristics between acute gastrointestinal (GI)-graft-versus-host disease (GVHD) and infectious colitis (IC) after hematopoietic stem cell transplantation (HSCT). Of the 282 patients who underwent HSCT at our institution between January 1991 and December 2015, we could investigate 182 patients in detail. Of the 182 patients, we selected those who underwent colonoscopy and were diagnosed with acute GI-GVHD or IC after HSCT. Patients' backgrounds, colonoscopic findings, and pathological findings were retrospectively analyzed. There were 30 patients who had colonoscopy performed and diagnosed with acute GI-GVHD or IC after HSCT. Of the 30 patients, 20 had acute GI-GVHD and 10 had IC. All the cases of acute GI-GVHD were diagnosed by endoscopic biopsy and 4 of the IC patients had Clostridium difficile associated colitis. In the IC group, the period from the transplantation up to diagnosis was significantly shorter than acute GI-GVHD group (10.0 days vs. 43.2 days, p = 0.03). In the acute GI-GVHD group, tortoiseshell-like mucosal patterns were significantly more common than the IC group (70% vs. 0%, p < 0.001). Furthermore, there were some cases presenting normal mucosal appearance despite the diagnosis with acute GI-GVHD by pathological findings. Clinically, we should consider IC when abdominal symptoms appeared in the early period after HSCT. Endoscopically, tortoiseshell-like mucosal pattern was a characteristic feature of acute GI-GVHD. In addition, it is essential to perform mucosal biopsy for diagnose of acute GI-GVHD even in patients showing the normal mucosal appearance.