Parkinson's disease (PD) is a debilitating neurodegenerative disorder causing severe motor disabilities resulting from the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) region of the midbrain. MicroRNAs (miRNAs) are small, non-coding RNAs which play a major role in several cellular processes in health and disease by regulating gene expression post-transcriptionally. Aberrant miRNA expression has been detected in post-mortem human PD brain samples, in vitro and in vivo PD models. However, none of the studies have focused on the role of the brain-abundant miR-124 in PD. In this study, we have evaluated the expression changes of miR-124 in the SN of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. MiRNA expression analysis by qPCR revealed a decrease in the expression of brain-enriched miR-124 in the SN of MPTP-treated mice as compared to controls. Further, in vitro study revealed a decrease in the expression of miR-124 in MN9D dopaminergic neurons treated with methyl phenyl pyridinium (MPP) iodide. The expression of calpains 1 and 2 which is modulated by miR-124 was increased in the SNc of MPTP-treated mice as observed at different time points after treatment and in the MN9D dopaminergic neurons treated with MPP iodide leading to increased expression of the p35 cleavage product, p25 and cyclin-dependent kinase 5 (cdk5). Calpain-p25-mediated increase in cdk5 expression leading to dopaminergic neuronal death has been demonstrated in human PD and MPTP-PD models. Increased expression of calpain 1/cdk5 pathway proteins was observed in anti-miR-124-transfected MN9D cells in our studies. Knockdown of miR-124 led to increased production of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) both known to increase oxidative stress. Further, experiments with miR-124 target protector sequences specific to calpain 1 revealed an interaction of miR-124 with calpain 1. Overexpression of miR-124 after MPP iodide treatment on MN9D cells was found to attenuate the expression of the calpain 1/p25/cdk5 proteins while improving cell survival. These results suggest that miR-124 acts to modulate the expression of calpain/cdk5 pathway proteins in the dopaminergic neurons. A better understanding of the mechanisms controlling the expression of miR-124 will aid in targeting miR-124 for better treatment strategies for PD.