Recent studies have suggested that 5-aminosalicylic acid (5-ASA) inhibits colorectal cancer (CRC) development. However, the mechanism underlying the antineoplastic effect of 5-ASA remains unknown. We here examined the effect of 5-ASA on epidermal growth factor receptor (EGFR) activation, a pathway that triggers mitogenic signals in CRC cells. We show that 5-ASA inhibits EGFR activation, through a mechanism that does not rely on CRC cell death induction. 5-ASA enhances the activity, but not expression, of phosphorylated (p)-EGFR-targeting phosphatases (PTPs), and treatment of cells with PTP inhibitors abrogates the 5-ASA-mediated EGFR dephosphorylation. Both SH-PTP1 and SH-PTP2 interact with EGFR upon 5-ASA treatment. However, knockdown of SH-PTP2 but not SH-PTP1 by small interference RNAs prevents the 5-ASA-induced EGFR dephosphorylation. Finally, we show that 5-ASA attenuates p-EGFR in ex vivo organ cultures of CRC explants. Data indicate that 5-ASA disrupts EGFR signalling by enhancing SH-PTP2 activity, and suggest a mechanism by which 5-ASA interferes with CRC growth.