Metastatic dissemination of primary tumors is responsible for 90% of colorectal cancer (CRC) deaths. The presence of positive lymph nodes, which separates stage I/II from stage III CRC, is a particularly key factor in patient management. Here, we describe results of a quantitative proteomic survey to identify molecular correlates of node status. Laser capture microdissection and two-dimensional difference gel electrophoresis were used to establish expression profiles for 980 discrete protein features in 24 human CRC specimens. Protein abundances were determined with a median technical coefficient of variation of 10%, which provided an ability to detect small differences between cancer subtypes. Transgelin, a 23-kDa actin-binding protein, emerged as a top-ranked candidate biomarker of node status. The area under the receiver operating characteristic curve for transgelin in predicting node status was 0.868 (P = .002). Significantly increased frequency of moderate- and high-level transgelin expression in node-positive CRC was also seen using semiquantitative immunohistochemistry to analyze 94 independent CRC specimens on tissue microarrays (P = .036). Follow-up studies in CRC cell lines demonstrated roles for transgelin in promoting invasion, survival, and resistance to anoikis. Transgelin localizes to the nucleus of CRC cells, and its sequence and properties suggest that it may participate in regulation of the transcriptional program associated with the epithelial-to-mesenchymal transition.