Ethyl pyruvate (EP) has been increasingly appreciated as an anti-inflammatory and neuroprotective agent with potent pharmacological properties relevant for treatment of various CNS disorders. Microglial cells seem to be particularly sensitive to its effects. In this study, microglial cells were exposed to EP for relatively short periods (10–120min) and inflammatory properties of the cells were determined after 24h of cultivation. Application of EP in the short-term periods inhibited production of interleukin-6, tumor necrosis factor and nitric oxide in microglial cells. At the same time, the effects on cell viability, reactive oxygen species generation and expression of F4/80 and CD40 of microglial cells were minor. NFκB activation was not affected by EP in the cells during the short exposures, thus implying that the observed effect of EP on cytokine and nitric oxide generation was performed in NFκB independent way. Importantly, effects of the short term EP treatment on microglial cells were detected by a real time cell analysis, as well. The observed ability of EP to affect microglial cell function after relatively short time of exposure is relevant for its therapeutic potential against inflammatory disorders of the CNS.