Altered extracellular matrix (ECM) remodeling is an important part of the pathology of gastrointestinal (GI) disorders. In the intestine, type XVI collagen (col-16) plays a role in pathogenesis by affecting ECM architecture and induce cell invasion. Measuring col-16 in serum may therefore have biomarker potential in GI disorders such as colorectal cancer (CRC) and ulcerative colitis (UC). The aim of this study was to determine whether col-16 can serve as a biomarker for altered ECM remodeling in patients with CRC and UC. A monoclonal antibody was raised against the C-terminal end of col-16 (PRO-C16), and a competitive enzyme-linked immunosorbent assay (ELISA) was developed and technically validated. Levels of PRO-C16 were measured in serum from patients with CRC (before (n = 50) and 3 months after (n = 23) tumor resections), UC (n = 39) and healthy controls (n = 50). The PRO-C16 ELISA was specific toward the C-terminal of col-16. PRO-C16 was significantly elevated both in serum from patients with CRC (P = 0.0026) and UC (P < 0.0001) compared to controls. No difference was detected in levels of PRO-C16 between patients with CRC at baseline and 3 months after tumor resections (P > 0.999). Levels of PRO-C16 identified patients with a GI disorder with a positive predictive value of 0.9 and an odds ratio of 12 (95%CI = 4.5-29.5, P < 0.0001). The newly developed assay detected significantly elevated levels of PRO-C16 in serum from patients with GI disorders compared to controls suggesting its potential as a biomarker in this setting. Future studies are needed to validate these findings.