The four human Argonaute (AGO) proteins, essential in RNA interference and gene regulation, exhibit high sequence and structural similarity, yet differ functionally. Our molecular dynamics simulations revealed that while AGO proteins adopt similar open-close states, each one displays distinct local conformations, leading to unique interdomain distances and intramolecular interactions. We found that GW182/ZSWIM8 interaction sites, catalytic/pseudo-catalytic tetrads and long common protein subsequences conserve their molecular movement with minute differences but have varying solvent accessibility per AGO. We observed diverse stability patterns at the post-transcriptional sites of the AGOs, except for AGO4. Combining simulation data with large datasets of experimental structures and AlphaFold’s predictions, we identified proteins with gene sequence and protein structure similarities that operate in the mitosis pathway and share mitosis-related interactors and miRNA targets. Additionally, we suggest a zinc ion binding function in AGO proteins, predicting potential binding sites and detecting structurally similar proteins with the same function.