Gliomas are the most aggressive adult primary brain tumors. Expression of inducible Nitric Oxide Synthase has been reported as a hallmark of chemoresistance in gliomas and several studies have reported that inhibition of inducible Nitric Oxide Synthase could be related to a decreased proliferation of glioma cells. The present work was to analyze the molecular effects of the acetamidine derivative compound 39 (formally CM544， -(3-{[(1-iminioethyl)amino]methyl}benzyl) prolinamide dihydrochloride)， a newly synthetized iNOS inhibitor， in a C6 rat glioma cell model. There is evidence of CM544 selective binding to the iNOS， an event that triggers the accumulation of ROS/RNS， the expression of Nrf-2 and the phosphorylation of MAPKs after 3 h of treatment. In the long run， CM544 leads to the dephosphorylation of p38 and to a massive cleavage of PARP-1， confirming the block of C6 rat glioma cell proliferation in the G1/S checkpoint and the occurrence of necrotic cell death.