Glypican-1 (GPC1) is a cell surface proteoglycan that is upregulated in multiple types of human cancers including pancreatic cancer. Here, we investigated whether GPC1 could be a target of antibody-toxin fusion proteins (i.e., immunotoxins) for treating pancreatic cancer. We constructed a panel of GPC1-targeted immunotoxins derived from a functional domain of Pseudomonas exotoxin A. An albumin-binding domain was also introduced into the anti-GPC1 immunotoxin to improve serum half-life. Small-molecule screening was performed to identify irinotecan that shows synergistic efficacy with the immunotoxin. We showed that GPC1 was internalized upon antibody binding. Anti-GPC1 immunotoxins alone inhibited tumor growth in a pancreatic cancer xenograft model. The immunotoxin treatment reduced active β-catenin expression in tumor cells. Furthermore, immunotoxins containing an albumin-binding domain in combination with irinotecan caused pancreatic tumor regression. GPC1 expression was reduced by the immunotoxin treatment due to the degradation of the internalized GPC1 and its short cellular turnover rate. Our data indicate that the GPC1-targeted immunotoxin inhibits pancreatic tumor growth via degradation of internalized GPC1, downregulation of Wnt signaling, and inhibition of protein synthesis. The anti-GPC1 immunotoxin in combination with irinotecan thus provides a potential new treatment strategy for patients with pancreatic tumors.