<p>RNA interference is a powerful gene regulatory strategy in which RNA molecules participate in the sequence-specific inhibition of gene expression by dsRNA, either through transcriptional or translational suppression. Since the RNA-mediated interference (RNAi) has been acknowledged as a natural method for controlling gene expression in higher species, ranging from plants to people, it has a stronger impact on crop improvement. In this review, we focused on additional research in numerous areas including microRNA (mi RNA), small interfering RNA (siRNA), plant RNAi transformation vector and transgenic RNAi plants, and off-target effects. The RNAi mechanism involves small interfering RNA molecules that efficiently down-regulate desired genes. Manipulating genes through small RNA interference can lead to improved traits in crops, such as resistance to biotic and abiotic stresses, as well as nutritional enhancement through bio-fortification. We also summarized the successful use of RNAi in altering several desired traits in plants such as changes in morphology, increased nutrients content, enhanced plant biomass and grain yield, extended shelf life, development of seedless fruit, induction of male sterility, enhanced synthesis of secondary metabolite, improved biofuel production and enhanced defense against biotic and abiotic stresses. This review article presents an outline of the ideas and mechanisms of the RNAi approach in crop improvement through gene regulation and explores various applications across diverse aspect of crop enhancement while addressing current challenges.</p>