Background/Aims: Ulcerative colitis (UC), along with Crohn’s disease, is one of the main types of inflammatory bowel disease (IBD). On the other hand, deregulated autophagy is involved in many chronic diseases, including IBD. In this study, we aimed to investigate the role of Atg5 and microRNA-181a (miR-181a) in the pathophysiology of UC. Methods: Colon biopsy, stool, and blood samples of 6 men and 9 women were confirmed for UC. Also, 13 men and 17 women were selected as healthy control (HC). Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used to measure the Atg-5 content of the colon biopsies. Besides, the serum and stool levels of Atg5 were measured using ELISA. Moreover, the total RNA of blood cells was extracted and evaluated for the expression of miR-181a. Results: We found 1.2 ng/mL versus 0.46 ng/mL, 0.34 ng/mL versus 0.24 ng/mL, and 0.082 ng/mL versus 0.062 ng/mL of Atg5 in stool, intestinal tissue, and serum of UC and HCs, respectively. There was no significant difference in the expression of miR-181a in the blood samples of UC and HCs. Immunohistochemistry showed high positivity without any significant difference between the 2 groups in the quantitative analysis. Conclusions The significant difference observed between the stool Atg5 content of the HCs and UC patients may provide new insight into using this protein as a diagnostic biomarker, however, considering the small size of our studied population further studies are needed.