MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by binding to target mRNAs with perfect or imperfect complementarity, recruiting an Argonaute (AGO) protein complex that usually results in degradation or translational repression of the target mRNA. AGO proteins function as the Slicer enzyme in miRNA and small interfering RNA (siRNA) pathways involved in human physiological and pathophysiological processes, such as antiviral responses and disease formation. Although the past decade has witnessed rapid advancement in studies of AGO protein functions, to further elucidate the molecular mechanism of AGO proteins in cellular function and biochemical process is really a challenging area for researchers. In order to understand the molecular causes underlying the pathological processes, we mainly focus on five fundamental problems of AGO proteins, including evolution, functional domain, subcellular location, post-translational modification and protein-protein interactions. Our discussion highlight their roles in early diagnosis, disease prevention, drug target identification, drug response, etc.