GABAB receptors are heterodimeric G protein-coupled receptors, which control neuronal excitability by mediating prolonged inhibition. The magnitude of GABAB receptor-mediated inhibition essentially depends on the amount of receptors in the plasma membrane. However, the factors regulating cell surface expression of GABAB receptors are poorly characterized. Cell surface GABAB receptors are constitutively internalized and either recycled to the plasma membrane or degraded in lysosomes. The signal that sorts GABAB receptors to lysosomes is currently unknown. Here we show that Mind bomb-2 (MIB2) mediated K63-linked ubiquitination of the GABAB1subunit at multiple sites is the lysosomal sorting signal for GABAB receptors. We found that inhibition of lysosomal activity in cultured rat cortical neurons increased the fraction of K63-linked ubiquitinated GABAB receptors and enhanced the expression of total as well as cell surface GABAB receptors. Mutational inactivation of four putative ubiquitination sites in theGABAB1 subunit significantly diminished K63-linked ubiquitination of GABAB receptors and prevented their lysosomal degradation. We identified MIB2 as the E3 ligase triggering K63-linked ubiquitination and lysosomal degradation of GABAB receptors. Finally, we show that sustained activation of glutamate receptors, a condition occurring in brain ischemia that downregulates GABAB receptors, considerably increased the expression of MIB2 and K63-linked ubiquitination of GABAB receptors. Interfering with K63-linked ubiquitination by overexpressing ubiquitin mutants or GABAB1 mutants deficient in K63-linked ubiquitination prevented glutamate-induced down-regulation of the receptors. These findings indicate that K63-linked ubiquitination of GABAB1 at multiple sites by MIB2 controls sorting of GABAB receptors to lysosomes for degradation under physiological and pathological condition.