Chemotherapy-induced mucositis is characterized by damage of mucous membranes throughout the alimentary tract (AT). Extracellular matrix (ECM) components play a vital role in maintaining mucosal barrier integrity by regulating cellular apoptosis, proliferation and differentiation of overlying epithelial cells. The aims of this study were to characterize the changes in epithelial cell kinetics and to investigate the expression of the ECM components in the gastrointestinal tract following irinotecan administration. Female dark agouti rats were treated with single 200 mg/kg dose irinotecan and killed at various time points (1, 6, 24, 48, 72, 96 and 144 h) after treatment. Ki67 immunostaining and TUNEL were used to assess proliferation and apoptosis, respectively, in the jejunum and colon. Masson trichrome staining and picro-sirius red staining were used to determine the level of collagen, and immunohistochemistry was used to further assess collagen IV, fibronectin and laminin 1 and 2 expression in these tissues. Irinotecan halved cellular proliferation in the jejunum and colon at 48 and 24 h, respectively, while apoptosis peaked at 6 h (P < 0.05). There was a substantial increase in total collagen deposits around crypts from 24 h in both regions. However, collagen IV expression decreased significantly in the crypt region in a delayed fashion (P < 0.05). Fibronectin expression decreased significantly in jejunum and colon from 6 to 24 h following treatment (P < 0.05). Irinotecan induced a significant alteration in epithelial cell kinetics in both the jejunum and colon, and this correlated with changes in ECM component expression. Changes in ECM expression may have a direct impact on the loss of mucosal layer integrity evident in chemotherapy-induced mucositis.