AbstractThe control of expression in genetic regulation is a fundamental process for cell life. In RNA‐mediated silencing, human Argonaute‐2 protein (hAgo2) uses sequence information encoded in small RNAs (guide) to identify complementary sites in messenger RNAs (target) for repression. The specificity of this molecular recognition lies at the basis of the mechanisms that control the expression of thousands of genes, which necessarily requires a fine tuning of complex events. Among these, the binding of the first nucleotide of the target RNA (t1) is emerging as an important modulator of hAgo2‐mediated machinery. Using atomistic molecular dynamics‐derived analyses, we address the mechanism behind t1‐dependent regulation and study the impact of different t1 nucleotides (t1A, t1C, t1G, t1U) on the conformational dynamics of both hAgo2 and guide–target RNAs. Only when an adenine is found at this position, t1 directly interacts with a specific hAgo2 binding pocket, favoring the stabilization of target binding. Our findings show that hAgo2 exploits a dynamic recognition mechanism of the t1‐target thanks to a modulation of RNA conformations. Here, t1‐adenine is the only nucleobase endowed with a dual binding mode: a T‐shape and a co‐planar conformation, respectively, orthogonal and parallel to the following base‐pairs of guide–target duplex. This triggers a composite set of molecular interactions that stabilizes distinctive conformational ensembles. Our comparative analyses show characteristic traits of local and global dynamic interplay between hAgo2 and the RNA molecules and highlight how t1A binding acts as a molecular switch for target recognition and complex stabilization. Implications for future mechanistic studies are discussed.