Dysregulation of the tightly controlled protein phosphorylation networks that govern cellular behavior causes cancer. The membrane-associated, intracellular protein tyrosine phosphatase PTP4A3 is overexpressed in human colorectal cancer and contributes to cell migration and invasion. To interrogate further the role of PTP4A3 in colorectal cancer cell migration and invasion, we deleted the Ptp4a3 gene from murine colorectal tumor cells. The resulting PTP4A3−/− cells exhibited impaired colony formation, spheroid formation, migration, and adherence compared with the paired PTP4A3fl/fl cells. We replicated these phenotypic changes using the new small-molecule, allosteric PTP4A3 inhibitor JMS-053. A related structure, JMS-038, which lacked phosphatase inhibition, displayed no cellular activity. Reduction in cell viability and colony formation by JMS-053 occurred in both mouse and human colorectal cell lines and required PTP4A3 expression. Ptp4a3 deletion increased the expression of extracellular matrix (ECM) and adhesion genes, including the tumor suppressor Emilin 1. JMS-053 also increased Emilin 1 gene expression. Moreover, The Cancer Genome Atlas genomic database revealed human colorectal tumors with high Ptp4a3 expression had low Emilin 1 expression. These chemical and biologic reagents reveal a previously unknown communication between the intracellular PTP4A3 phosphatase and the ECM and support efforts to pharmacologically target PTP4A3.—McQueeney, K. E., Salamoun, J. M., Ahn J. G., Pekic, P., Blanco, I. K., Struckman, H. L., Sharlow, E. R., Wipf, P., Lazo, J. S. A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.