SNAT3 is a major facilitator of glutamine (Gln) efflux from astrocytes, supplying Gln to neurons for neurotransmitter synthesis. Our previous investigations have shown that, in primary cortical astrocyte cultures, SNAT3 protein is degraded after exposure to manganese (Mn(2+)). The present studies were performed to identify the processes responsible for this effect. One of the well-established mechanisms for protein-level regulation is posttranslational modification via ubiquitination, which leads to the rapid degradation of proteins by the 26S proteasome pathway. Here, we show that astrocytic SNAT3 directly interacts with the ubiquitin ligase, Nedd4-2 (neural precursor cells expressed developmentally downregulated 4-2), and that Mn(2+) increases both Nedd4-2 mRNA and protein levels. Additionally, we have found that Mn(2+) exposure elevates astrocytic ubiquitin B mRNA expression, free ubiquitin protein levels, and total protein ubiquitination. Furthermore, Mn(2+) effectively decreases astrocytic mRNA expression and the phosphorylation of serum and glucocorticoid-inducible kinase, a regulatory protein, which, in the active phosphorylated form, is responsible for the phosphorylation and subsequent inactivation of Nedd4-2. Additional findings establish that Mn(2+) increases astrocytic caspase-like proteolytic proteasome activity and that the Mn(2+)-dependent degradation of SNAT3 protein is blocked by the proteasome inhibitors, N-acetyl-leu-leu-norleucinal and lactacystin. Combined, these results demonstrate that Mn(2+)-induced SNAT3 protein degradation and the dysregulation of Gln homeostasis in primary astrocyte cultures proceeds through the ubiquitin-mediated proteolytic system.