Lymph node metastasis is an important factor determining outcome from colorectal cancer (CRC). Identification of molecular markers useful to predict lymph node metastasis is urgently needed. Our objective was to identify genes useful for characterization and prediction of lymph node metastasis in CRC. Gene expression profiles of cancer were determined by human U133 Plus 2.0 GeneChip® in 24 CRC patients, and patients with and without lymph node metastasis were compared. We focused on the visinin-like protein-1 (VSNL-1) gene and evaluated VSNL-1 mRNA expression levels with reverse transcriptase-polymerase chain reaction and immunohistochemical methods. Immunohistochemical evaluation of VSNL-1 mRNA expression was also performed in 143 other CRC patients to determine clinicopathological significance of VSNL-1. Twenty-four novel discriminating genes showed expression significantly different between patients with and without lymph node metastasis. Mean level of VSNL-1 mRNA expression in tumor tissue with lymph node metastasis was significantly higher than that in tissue without lymph node metastasis. Immunohistochemical examination demonstrated immunoreactivity for VSNL-1 in cytoplasm of the cancer cells with lymph node metastasis. High VSNL-1 expression was significantly associated with lymphatic invasion in stage II disease (p = 0.0061) and number of lymph node metastases in stage III disease (p = 0.0461). Patients with high VSNL-1 expression had significantly poorer prognosis than those with low expression in stage III disease (p = 0.045). This study is the first to demonstrate a prognostic role for VSNL-1 at the mRNA level, suggesting the possible usefulness of VSNL-1 as a predictor of lymph node metastasis and poor prognosis in CRC.