Pyruvate carboxylase (PC) is an enzyme catalyzing the conversion of pyruvate to oxaloacetate, which possesses anaplerotic role in cellular metabolism. The expression of PC was confirmed in cells of several cancer types, in which it ensures several cellular functions, such as growth and division. To investigate the expression of PC in human astrocytoma, glioblastoma and neuroblastoma cells we applied the immunodetection methods. The results of the Western blot analysis and immunocytochemical detection revealed the presence of PC in human astrocytoma, glioblastoma and neuroblastoma cells. Furthermore, application of PC inhibitor, 3-chloro-1,2-dihydroxypropane (CDP), negatively impacts the viability of astrocytoma cells. The cytotoxic effect of CDP could be partially reversed by application of citrate, 2-oxoglutarate and malate in incubation media. Our results revealed that astrocytoma, glioblastoma and neuroblastoma cells are equipped with PC, which might significantly contribute by its anaplerotic activity to sustain the metabolism of cancer cells.