The precise mechanism responsible for the frequent overexpression of cyclinD1 in human head and neck squamous cell carcinoma (HNSCC) is not known. In view of the fact that signal transducers and activators of transcription 3 (Stat3) is often activated in HNSCC cells, we examined the effects of Stat3 on cyclin D1 expression and cell proliferation in the YCU-H891 HNSCC cell line that displays constitutive activation of Stat3. Expression of a dominant negative Stat3 construct in YCU-H891 cells inhibited proliferation, cyclin D1 promoter activity, and cellular levels of cyclin D1 mRNA and protein. The levels of the antiapoptotic Bcl-2 and Bcl-X(L) proteins were also inhibited. In 51 primary tumor samples from patients with squamous cell carcinoma of the p.o. tongue, there was a significant correlation between increased levels of the activated form of Stat3, phosphorylated-Stat3, and increased levels of cyclin D1 (P < 0.0001). Increased tumor levels of phosphorylated-Stat3 were also associated with lower survival rates (P < 0.01). This study provides the first evidence that in HNSCC, constitutive activation of Stat3 plays a causative role in overexpression of cyclin D1, and in clinical studies, Stat3 activation may provide a novel prognostic factor. Furthermore, agents that target Stat3 may be useful in the treatment of HNSCC.