We investigated the role of NADPH oxidase (Nox) 4 in colon cancer development. The expression of Nox4, unlike Nox1, was specifically induced in colon carcinoma and was quite similar to that of transforming growth factor-β (TGF-β)1. TGF-β1 treatment of colon cancer cells upregulated the Nox4 expression and ROS production through SMAD3. Knockdown of Nox4 suppressed TGF-β1-induced cell motility. Nox4 modulated the Rho activity through redox regulation of the low molecular weight protein tyrosine phosphatase (LMW-PTP)-p190RhoGAP pathway upon TGF-β1 stimulation. These finding suggests that Nox4 contributes to TGF-β1-dependent motility of colon carcinoma through the LMW-PTP-p190RhoGAP-Rho signaling pathway.