Cardiolipin (CL) is a mitochondrial phospholipid, which has a well-defined role in regulating metabolic processes and maintaining homeostasis. While CL is normally confined to the inner mitochondrial membrane, it may be released extracellularly from damaged or dying cells during physiological and pathological conditions characterized by cellular stress, tissue damage, and cell death. Once released, CL can act as a signalling molecule by interacting with cells in an autocrine and paracrine manner. Previous research demonstrates that extracellular CL regulates select immune functions of microglia, which are the innate immune cells of the brain. However, the effects of CL on astrocyte functions are unknown. Astrocytes support metabolic homeostasis of neurons, facilitate neurotransmission, and are also active participants in neuroimmune responses of the brain. During chronic neuroinflammatory states, astrocytes become over-activated and may cause damage to the surrounding tissues and cells. We hypothesized that extracellular CL modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity. Therefore, we studied the effects of CL added to the culture media of primary murine astrocytes and human U118 MG astrocytic cells. The phagocytic activity of primary murine astrocytes was measured using fluorescent latex microspheres. The secretion of inflammatory cytokines was monitored by the enzyme-linked immunosorbent assay or immunoblotting. The expression of glial fibrillary acidic protein (GFAP) was measured using immunoblotting. Extracellular CL alone upregulated the phagocytic activity of primary murine astrocytes, as well as the release of monocyte chemoattractant protein (MCP)-1 and interferon (IFN)-β by human U118 MG astrocytic cells. CL inhibited the lipopolysaccharide (LPS)-induced increase in GFAP expression by U118 MG astrocytic cells, as well as the secretion of cytotoxins by the same cell type. Our study demonstrates that extracellular CL regulates select functions of astrocytes, which become dysregulated in chronic neuroinflammatory states, such as those observed in neurodegenerative diseases.