MicroRNAs (miRNAs), a class of small non-coding RNA molecules that mediate RNA interference (RNAi), have emerged as a promising alternative strategy for controlling spider mites. As a major pest mite threatening the citrus industry, Panonychus citri has caused substantial economic losses primarily due to its rapid evolution of resistance to conventional acaricides. Against this backdrop, the present study is designed to clarify the regulatory role of miRNAs in the eclosion process of P. citri and further develop transgenic miRNA-expressing soybean lines with enhanced resistance to this pest mite. To elucidate the regulatory mechanism of miRNAs in eclosion, a combination of miRNA overexpression, bioinformatics analysis, dual-luciferase activity assays, and RNAi assays was employed. For the generation of single-copy transgenic miRNA soybean lines with high transcript levels, Agrobacterium-mediated genetic transformation was adopted, coupled with PCR detection, miRNA expression profiling, and southern blot hybridization for verification. Additionally, detached leaf and whole-plant feeding bioassays were carried out to assess the control efficacy of transgenic soybeans against P. citri, while direct feeding and trophic transfer assays were performed to evaluate their safety to Neoseiulus barkeri. miR-750 and miR-200a-5p were identified as key miRNAs involved in the deutonymph-to-adult transition of P. citri. Specifically, miR-750 was found to bind to the target genes NFYB and ADRM1, while miR-200a-5p targeted ADRM1. Silence of NFYB or ADRM1via oral dsRNA led to eclosion abnormalities in P. citri, consistent with that in the overexpression of two miRNAs, Furthermore, We generated 18 and 17 soybean transformants expressing miR- 750 and miR-200a-5p, respectively, and identified two single-copy miR-750 lines with high transcript abundance and four miR-200a-5p lines. Phenotypic analysis of the T1 and T2 generations of these transgenic soybeans showed that both lines (2#14 for miR-750 and 7#6 for miR-200a-5p) exhibited high resistance to P. citri, and this resistance trait was stably inherited across the two generations, confirming its heritable stability. Notably, both lines (2#14 and 7#6) had adverse effects on Neoseiulus barkeri, a non-target predatory mite, indicating good ecological safety. Collectively, These findings highlight endogenous miRNAs as promising candidate targets for the development of environment-friendly pest mite control strategies, providing a novel and sustainable approach for managing P. citri.