MicroRNAs (miRNAs) regulate gene expression by guiding Argonaute (Ago) containing RNA-induced silencing complex (RISC) to specific target mRNA molecules. It is well established that miRNAs are stabilized by Ago proteins, but the molecular features that triggers miRNA destabilization from Ago proteins remain largely unknown. To explore the molecular mechanisms of how targets affect the stability of miRNAs in human Ago (hAgo) proteins, we employed an in vitro system that consists of a minimal hAgo2-RISC in HEK293T cell lysates. Surprisingly, we found that miRNAs are drastically destabilized by binding to seedless, non-canonical targets. We showed that miRNAs are destabilized at their 3ʹ ends during this process, which is largely attributed to the conformational flexibility of the L1-PAZ domain. Based on these results, we propose that non-canonical targets may play an important regulatory role in controlling the stability of miRNAs, instead of being regulated by miRNAs.