Neurotoxicity of individual metals is well investigated but that of metal mixture (MM), an environmental reality, in the developing brain is relatively obscure. We investigated the combinatorial effect of arsenic (As), cadmium (Cd), and lead (Pb) on rat brain development, spanning in utero to postnatal development. MM was administered by gavage to pregnant and lactating rats, and to postweaning pups till 2 months. The pups exhibited behavioral disturbances characterized by hyperlocomotion, increased grip strength, and learning-memory deficit. Disruption of the blood-brain barrier (BBB) was associated with dose-dependent increase in deposition of the metals in developing brain. Astrocytes were affected by MM treatment as evident from their reduced density, area, perimeter, compactness, and number of processes, and increased apoptosis in cerebral cortex and cerebellum. The metals induced synergistic reduction in glial fibrillary acidic protein (GFAP) expression during brain development; however, postweaning withdrawal of MM partially restored the levels of GFAP in adults. To characterize the toxic mechanism, we treated rat primary astrocytes with MM at concentrations ranging from lethal concentration (LC)(10) to LC(75) of the metals. We observed synergistic downregulation in viability and increase in apoptosis of the astrocytes, which were induced by proximal activation of extra cellular signal-regulated kinase (ERK) signaling and downstream activation of Jun N-terminal kinase (JNK) pathway. Furthermore, rise in intracellular calcium ion ([Ca(2+)](i)) and reactive oxygen species generation promoted apoptosis in the astrocytes. Taken together, these observations are the first to show that mixture of As, Cd, and Pb has the capacity to induce synergistic toxicity in astrocytes that may compromise the BBB and may cause behavioral dysfunction in developing rats.