Astrocytes, the predominant glial cells in the central nervous system (CNS), play diverse roles including metabolic support for neurons, provision of neurotrophic factors, facilitation of synaptic neurotransmitter uptake, regulation of ion balance, and involvement in synaptic formation. The accumulation of lipids has been noted in various neurological conditions, yet the response of astrocytes to lipid-rich environments remains unclear. In this study, primary astrocytes isolated from the neonatal rat cortex were exposed to a lipid mixture (LM) comprising cholesterol and various fatty acids to explore their reaction. Our results showed that astrocyte viability remained unchanged following 24 h of 5% or 10% LM treatment. However, exposure to LM for 96 h resulted in reduced cell viability. In addition, LM treatment led to the accumulation of lipid droplets (LDs) in astrocytes, with LD size increasing over prolonged exposure periods. Following 24 h of LM treatment and then 48 h in fresh medium, a significant reduction in intracellular LD size was observed in cultures treated with 5% LM, while no change occurred in cultures exposed to 10% LM. Yet, exposure to 10% LM for 24 h significantly increased the expression of the cholesterol efflux regulatory protein/ATP-binding cassette transporter (ABCA1) gene, responsible for intracellular cholesterol efflux, resulting in reduced cholesterol content within astrocytes. Moreover, LM exposure led to decreased mitochondrial membrane potential (MMP) and increased levels of mature apoptosis-inducing factor (AIF). The smaller LDs were observed to co-localize with microtubule-associated protein 1A/1 B light chain 3 B (LC3) and lysosomal-associated membrane protein-1 (LAMP-1) in LM-treated astrocytes, coinciding with lysosomal acidification. These results indicate that the continuous buildup of LDs in astrocytes residing in lipid-enriched environments may be attributed to disruptions caused by LM in mitochondrial and lysosomal functions. Such disruptions could potentially impede the supportive role of astrocytes in neuronal function.