Parkinson's disease (PD) is the most common neurodegenerative movement disorder, resulting in dopaminergic (DA) neuronal loss in the substantia nigra pars compacta (SNpc) and damage to the extranigral spinal cord neurons. Current therapies do not prevent the disease progression. Hence, developing efficacious therapeutic strategies for treatment of PD is of utmost importance. The goal of this study is to delineate the involvement of calpain-mediated inflammation and neurodegeneration in SN and spinal cord in MPTP-induced parkinsonian mice (C57BL/6 N), thereby elucidating potential therapeutic target(s). Increased calpain expression was found localized to tyrosine hydroxylase (TH(+)) neurons in SN with significantly increased TUNEL-positive neurons in SN and spinal cord neurons in MPTP mice. Inflammatory markers Cox-2, caspase-1, and NOS-2 were significantly upregulated in MPTP mouse spinal cord as compared to control. These parameters correlated with the activation of astrocytes, microglia, infiltration of CD4(+)/CD8(+) T cells, and macrophages. We found that subpopulations of CD4(+) cells (Th1 and Tregs) were differentially expanded in MPTP mice, which could be regulated by inhibition of calpain with the potent inhibitor calpeptin. Pretreatment with calpeptin (25 μg/kg, i.p.) attenuated glial activation, T cell infiltration, nigral dopaminergic degeneration in SN, and neuronal death in spinal cord. Importantly, calpeptin ameliorated MPTP-induced altered gait parameters (e.g., reduced stride length and increased stride frequency) as demonstrated by analyses of spatiotemporal gait indices using ventral plane videography. These findings suggest that calpain plays a pivotal role in MPTP-induced nigral and extranigral neurodegenerative processes and may be a valid therapeutic target in PD.