Aim:Glioblastoma multiforme (GBM), a primary brain tumor, is the most common, diffuse, highly invasive, and malignant type of brain tumor. c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family and play fundamental roles in signal transduction. The aim of this study was to evaluate the effects of JNK inhibition and signal blocking on hypoxia (hypoxia-inducible factor 1-alpha [HIF-1α]), differentiation and neurogenesis (bone morphogenetic protein [BMP4]), and the cytoskeleton (F-actin) in GBM cells (GBMCs). Material and Methods:We evaluated the differences between GBMCs and astrocytes in terms of the abovementioned parameters and assessed them with the aim of studying human GBMCs (U-87 MG) and astrocytes (SVGp12). The cells were exposed to different doses of the JNK inhibitor, SP600125, for 24, 48, and 72 h. HIF-1α, BMP4, and F-actin expressions were evaluated using immunofluorescence image analysis. Results:The half-maximal inhibitory concentration value for SP600125 was determined to be 10 μM at 24 h of exposure. After SP600125 administration, elevated levels of HIF-1α and BMP4 were detected in GBMCs and astrocytes. F-actin level only increased in GBMCs after SP600125 administration.Conclusion:JNKs are important for cell proliferation, differentiation, survival, and death; thus, research on JNKs has become important for the treatment of many human diseases, especially brain tumors, Parkinson’s disease, and Alzheimer’s disease. The results of this study involving immunofluorescence techniques should be investigated and supported by studies that involve comprehensive molecular techniques.