Although the mechanism of action of tissue‐type plasminogen activator (tPA) in excitotoxic necrosis is well documented, whether this serine protease can influence the apoptotic cascade remains a subject of debate. Here, we report that tPA protects cultured cortical neurones against apoptotic cell death induced by serum deprivation, an effect associated with a reduction of caspase‐3 activation. Interestingly, blocking tPA proteolytic activity by either tPA stop or neuroserpin did not prevent this neuroprotection. Similarly, prevention of the interaction between tPA and its receptor low‐density lipoprotein receptor‐related protein (LRP) could not alter tPA anti‐apoptotic activity. Interestingly, the survival‐promoting effect of tPA was blocked by the phosphatidylinositol‐3 (PI‐3) kinase inhibitor, LY294002, but not by the mitogen‐activated protein (MAP) kinase inhibitor, U0126. In conclusion, the present demonstration of an anti‐apoptotic effect of tPA, independent of its enzymatic activity, reveals an additional level of complexity in our understanding of this critical mediator of brain physiology and pathology.