Expression of the tumor suppressor deleted in liver cancer‐1 (DLC‐1) is lost in non‐small cell lung (NSCLC) and other human carcinomas, and ectopic DLC‐1 expression dramatically reduces proliferation and tumorigenicity. DLC‐1 is a multi‐domain protein that includes a Rho GTPase activating protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC‐1 in tumor suppression, we performed biochemical and biological studies evaluating DLC‐1 in NSCLC. Full‐length DLC‐1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5‐ to 20‐fold enhanced activity for RhoA, RhoB, RhoC, and Cdc42. DLC‐1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC‐1 expression in DLC‐1‐deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC‐1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC‐1 expression in NSCLC cell lines impaired both anchorage‐dependent and ‐independent growth, as well as invasion in vitro. Surprisingly, we found that the anti‐tumor activity of DLC‐1 was due to both RhoGAP‐dependent and ‐independent activities. Unlike the rat homologue p122RhoGAP, DLC‐1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C‐δ1. Combined, these studies provide information on the mechanism of DLC‐1 function and regulation, and further support the role of DLC‐1 tumor suppression in NSCLC. © 2007 Wiley‐Liss, Inc.