Abstract MicroRNAs (miRNAs) are essential regulators of gene expression, defined by their unique biogenesis, which requires the precise excision of the small RNA from an imperfect fold-back precursor. Unlike their animal counterparts, plant miRNA precursors exhibit variations in sizes and shapes. Plant MIRNAs can undergo processing in a base-to-loop or loop-to-base direction, with DICER-LIKE1 (DCL1) releasing the miRNA after two cuts (two-step MIRNAs) or more (sequential MIRNAs). In this study, we demonstrate the critical role of the miRNA/miRNA* duplex region in the processing of miRNA precursors. We observed that endogenous MIRNAs frequently experience suboptimal processing in vivo due to mismatches in the miRNA/miRNA* duplex, a key region that fine-tunes miRNA levels. Enhancing the interaction energy of the miRNA/miRNA* duplex in two-step MIRNAs results in a substantial increase in miRNA levels. Conversely, sequential MIRNAs display distinct and specific requirements for the miRNA/miRNA* duplexes along their foldback structure. Our work establishes a connection between the miRNA/miRNA* structure and precursor processing mechanisms. Furthermore, we reveal a link between the biological function of miRNAs and the processing mechanism of their precursors with the evolution of plant miRNA/miRNA* duplex structures.