The article provides a comprehensive overview of the molecular mechanisms underlying RNA interference (RNAi), focusing on the roles of small regulatory RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). RNAi is a widespread process in eukaryotes that regulates gene expression during development and in response to stresses, including viral infection. The key steps in the RNAi pathway involve the production and action of small regulatory RNAs, which are governed by specialized ribonucleases and RNA binding proteins. Pre-miRNAs are initially processed in the nucleus by Drosha, a microprocessor complex comprising Drosha and DGCR8, into pre-miRNAs. These pre-miRNAs are then transported to the cytoplasm, where Dicer cleaves them into mature miRNAs and siRNAs. These double-stranded products assemble with Argonaute proteins, forming the RNA-induced silencing complex (RISC). One strand of the duplex is preferentially selected and used to guide sequence-specific silencing of complementary target mRNAs through endonucleolytic cleavage or translational repression. The article discusses the structural and mechanistic studies of the human miRNA pathway, including the roles of key proteins such as Drosha, Dicer, and Argonaute. Drosha and DGCR8 form the microprocessor complex, which cleaves pri-miRNAs into pre-miRNAs. Dicer generates dsRNAs suitable for loading onto Argonaute proteins, and Argonaute proteins recognize and bind to complementary target mRNAs, leading to silencing. The article also explores the interactions between these proteins, such as the binding of Dicer to Argonaute and the role of dsRNA-binding proteins (dsRBPs) in strand selection and RISC loading. Recent advances in structural biology have provided insights into the molecular mechanisms of RNAi, including the recognition of guide strand termini by Argonaute proteins and the conformational changes that occur upon target binding. Finally, the article highlights ongoing challenges and future directions in the field, such as the mechanisms of strand selection, the kinetics of repression and decay, and the role of P-bodies in RNA-driven silencing.The article provides a comprehensive overview of the molecular mechanisms underlying RNA interference (RNAi), focusing on the roles of small regulatory RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). RNAi is a widespread process in eukaryotes that regulates gene expression during development and in response to stresses, including viral infection. The key steps in the RNAi pathway involve the production and action of small regulatory RNAs, which are governed by specialized ribonucleases and RNA binding proteins. Pre-miRNAs are initially processed in the nucleus by Drosha, a microprocessor complex comprising Drosha and DGCR8, into pre-miRNAs. These pre-miRNAs are then transported to the cytoplasm, where Dicer cleaves them into mature miRNAs and siRNAs. These double-stranded products assemble with Argonaute proteins, forming the RNA-induced silencing complex (RISC). One strand of the duplex is preferentially selected and used to guide sequence-specific silencing of complementary target mRNAs through endonucleolytic cleavage or translational repression. The article discusses the structural and mechanistic studies of the human miRNA pathway, including the roles of key proteins such as Drosha, Dicer, and Argonaute. Drosha and DGCR8 form the microprocessor complex, which cleaves pri-miRNAs into pre-miRNAs. Dicer generates dsRNAs suitable for loading onto Argonaute proteins, and Argonaute proteins recognize and bind to complementary target mRNAs, leading to silencing. The article also explores the interactions between these proteins, such as the binding of Dicer to Argonaute and the role of dsRNA-binding proteins (dsRBPs) in strand selection and RISC loading. Recent advances in structural biology have provided insights into the molecular mechanisms of RNAi, including the recognition of guide strand termini by Argonaute proteins and the conformational changes that occur upon target binding. Finally, the article highlights ongoing challenges and future directions in the field, such as the mechanisms of strand selection, the kinetics of repression and decay, and the role of P-bodies in RNA-driven silencing.