Research states that Altered synaptic plasticity is often associated with major depressive disorder (MDD). Disease-associated changes in synaptic functions are tightly correlated with altered microRNA (miRNA) expression. Here, we examined the role of miRNAs and their functioning at the synapse in MDD by examining miRNA processing machinery at synapse and sequencing miRNAs and analyzing their functions in synaptic and total tissue fractions obtained from dorsolateral prefrontal cortex (dlPFC) of 15 MDD and 15 matched non-psychiatric control subjects. A total of 333 miRNAs were reliably detected in the total tissue fraction. Out of 351 miRNAs reliably expressed in the synaptic fraction, 24 were uniquely expressed at synapse. In vitro transfection studies and gene ontology revealed involvement of these altered miRNAs in synaptic plasticity, nervous system development, and neurogenesis. Our findings provide new insights into the understanding of the regulation of miRNAs at the synapse and their possible roles in MDD pathogenesis. Major depressive disorder (MDD) is one of the most debilitating mental disorders worldwide with a lifetime prevalence of 10.8% [1]. In conclusion, our study, for the first time, shows that a large number of miRNAs are synaptically enriched and a pool of miRNAs are uniquely associated with synapse. These synaptic miRNAs are differentially regulated in MDD subjects. In addition, there is a shift in the expression of synaptically enriched miRNAs, suggesting that miRNAs may be processed locally at synapse and this processing may be aberrant in MDD brain.