The c-Jun NH2-terminal kinase (JNK), which belongs to mitogen-activated protein kinase family, plays a major role in apoptosis in various cell types. JNK activation, however, also contributes to proliferation, survival, and tumorigenesis in some tumors, including gliomas. In this study, we used an immunohistochemical approach to examine the activation status of JNK of 226 gliomas in a high-density tissue microarray comprising all WHO codified WHO diffuse glioma subtypes and grades. The results were correlated with grade and EGFR expression status. Constitutively activated JNK (pJNK) was detected in 90.5%, 62.9% and 17.5% of WHO grade IV, III and II gliomas, respectively (p < 0.001). pJNK expression was not detected in the astrocytes or oligodendrocytes of any of 10 normal cerebral and cerebellar brain tissue samples. Among the 76 diffuse gliomas that exhibited EGFR expression, 63 (82.9%) were positive for pJNK. In contrast, only 50% (36/72) of the gliomas that were negative for EGFR were positive for pJNK (p < 0.0001). Overexpression of EGFR vIII in U87 cells or EGF treatment of U87-EGFR stable cells led to marked increase in JNK activation compared to parental U87 cells. Our data thus provide strong support for the hypothesis that JNK activation plays a role in the tumorigenesis and/or progression of diffuse gliomas, and suggests that EGFR is involved in constitutive JNK activation in diffuse gliomas. The ability to inhibit JNK activation might confer increased sensitivity to therapeutic modalities targeting this pathway.