In colorectal cancer there is a need for molecular markers that can complement the histopathological staging in predicting the likelihood of disease recurrence following curatively intended surgery. B7-H3 is an immunoregulatory protein shown to be overexpressed in several cancer forms, often associated with more advanced disease and poor prognosis. We wanted to examine whether B7-H3 could be a potential prognostic marker in colorectal cancer. Paraffin-embedded samples from 277 colorectal cancer patients were immunostained with anti-B7-H3 antibody. B7-H3 was expressed in the tumor cell cytoplasm and cell membrane in 62% and 46% of the samples, respectively. Unexpectedly, B7-H3 was expressed in the nucleus in 30% of the tumors. The nuclear localization was confirmed by Western immunoblotting of subcellular fractions. Importantly, in colon cancer, nuclear B7-H3 expression was independently and significantly associated with reduced metastasis-free, disease-specific and overall survival. B7-H3 expression in tumor-associated vasculature and fibroblasts was observed in the majority of samples, and endothelial B7-H3 expression was also significantly associated with poor outcome in colon cancer. In rectal cancer patients, the only significant association was between fibroblast B7-H3 expression and shorter metastasis-free survival. Few significant associations to clinicopathological parameters were seen. The results indicate that nuclear B7-H3 might be involved in colon cancer progression and metastasis, and suggest that nuclear B7-H3 could become a useful prognostic marker in colon cancer.