The cysteine carboxypeptidase cathepsin X is an important player in degenerative processes under normal ageing and pathological conditions. In the present study, we investigated the potential role of cathepsin X in 6-hydroxydopamine (6-OHDA)-induced toxicity in the pheochromocytoma cell line PC12 and neuroblastoma cell line SH-SY5Y. Cells exposed to 6-OHDA demonstrated alterations in the protein level of cathepsin X and activity of cathepsin X. Downregulation of cathepsin X expression by siRNA attenuated the neuronal death caused by 6-OHDA. Treatment with specific cathepsin X inhibitor AMS36 protected cells against 6-OHDA mediated cytotoxicity, resulting in reduced cell death and apoptosis. Furthermore, AMS36 reversed 6-OHDA-induced loss of tyrosine hydroxylase and attenuated 6-OHDA-induced activation of caspase-3, triggering apoptosis, intracellular generation of reactive oxygen species and mitochondrial dysfunction, including the release of cytochrome c and an imbalanced Bax/Bcl-2 ratio. Moreover, AMS36 interfered with NF-κB activation by blocking degradation of IκBα, preventing NF-κB translocation to the nucleus. Our data provide the first evidence that inhibition of cathepsin X protects both, PC12 and SH-SY5Y cells against 6-OHDA toxicity and indicate that cathepsin X may be responsible for dopamine neuron death, involved in the pathogenic cascade event for the neurodegenerative disorders, such as Parkinson's disease.