Gastrointestinal stromal tumors are the most common mesenchymal tumors found in the gastrointestinal tract. Their biological behavior is still predicted by a consensus scheme proposed by the U.S. National Institutes of Health. In this study, we investigated the prognostic significance of p16 protein expression in gastrointestinal stromal tumors. Expression of p16 protein was observed in 42.4% (92/217) of tumors and was significantly associated with a high mitotic count, tumor necrosis, recurrence or metastasis, and a higher-risk group. Patients with p16 expressing gastrointestinal stromal tumors showed a shorter overall survival and disease-free survival than those without p16 expression; however, p16 expression was not an independent prognostic factor. The risk of malignant behavior and the presence of recurrence or metastasis were independent prognostic factors. Expression of p16 protein predicts poor outcome and can be a useful marker to predict relapse or metastasis and aggressive behavior in gastrointestinal stromal tumors.