GPA33 is a promising surface antigen for targeted therapy in colorectal cancer (CRC). It is expressed almost exclusively in CRC and intestinal epithelia. However, previous clinical studies have not achieved expected response rates. We investigated GPA33 expression and regulation in CRC and developed a GPA33-targeted cellular therapy. We examined GPA33 expression in CRC cohorts using immunohistochemistry and immunofluorescence. We analyzed GPA33 regulation by interference with oncogenic signaling in vitro and in vivo using inhibitors and conditional inducible regulators. Furthermore, we engineered anti-GPA33-CAR T cells and assessed their activity in vitro and in vivo. GPA33 expression showed consistent intratumoral heterogeneity in CRC with antigen loss at the infiltrative tumor edge. This pattern was preserved at metastatic sites. GPA33-positive cells had a differentiated phenotype and low WNT activity. Low GPA33 expression levels were linked to tumor progression in patients with CRC. Downregulation of WNT activity induced GPA33 expression in vitro and in GPA33-negative tumor cell subpopulations in xenografts. GPA33-CAR T cells were activated in response to GPA33 and reduced xenograft growth in mice after intratumoral application. GPA33-targeted therapy may be improved by simultaneous WNT inhibition to enhance GPA33 expression. Furthermore, GPA33 is a promising target for cellular immunotherapy in CRC.