Both cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2) have been implicated in pathology of cerebral ischemia. However, which of PLA2 isoforms in astrocytes is responsible for arachidonic acid (AA) release contributing to their ischemic injury remains to be determined. The aim of the present study was to investigate the time-dependent activation of cPLA2 and sPLA2 in astrocytes exposed to combined oxygen glucose deprivation (OGD) as well as to evaluate the effectiveness of their pharmacological blockage as a method of preventing ischemic damage of the glial cells. It was shown that exposure of cultured astrocytes to OGD (0.5–24 h) causes an increase in cPLA2 and sPLA2 expression and activity. The role of AA liberated mainly by cPLA2 in the process of apoptosis was also demonstrated. To confirm the specific role of cPLA2 and sPLA2 in the mechanism of cells injury by OGD exposure, the effect of AACOCF3 as cPLA2 inhibitor and 12-epi-scalaradial as sPLA2 inhibitor on AA release was examined. It was proved that simultaneous pharmacological blockade of enzymatic activity of cPLA2 and sPLA2 during OGD by AACOCF3 and 12-epi-scalaradial substantially improves survival of ischemic injured glial cells.