Regulation of gene expression is a fundamental process in both prokaryotic and eukaryotic organisms. Multiple regulatory mechanisms are in place to control gene expression at the level of transcription, post-transcription and post-translation to maintain optimal RNA and protein expressions in cells. miRNAs (microRNAs) are abundant short 21–23 nt non-coding RNAs that are key regulators of virtually all eukaryotic biological processes. The levels of miRNAs in an organism are crucial for proper development and sustaining optimal cell functions. Therefore the processing and regulation of the processing of these miRNAs are critical. In the present chapter we highlight the most important steps of miRNA processing, describe the functions of key proteins involved in the maturation of miRNAs, and discuss how the generation and the stability of miRNAs are regulated.