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MicroRNAs (miRNAs) play essential roles in regulating various development processes in plants. However, their role in regulating rubber biosynthesis in <i>Eucommia ulmoides</i> is largely unknown. Rubber is mainly distributed in the tissue covering the seed (GZ) rather than the periphery (GB) of the pericarp of <i>E. ulmoides</i> during accumulation in May. To investigate the roles of miRNAs in rubber biosynthesis, we conducted high-throughput small RNA sequencing using GZ and GB collected on 11 May (rapid accumulation) and 11 June (reduced accumulation). In total, 12 and 25 miRNAs were either up- or downregulated in GZ in May (GZ511) compared to GB (GB511) in May, while 27 and 38 miRNAs were either up- or downregulated in GZ in May compared to GZ in June (GZ611), respectively. Functional analyses of differentially expressed (DE−) genes targeted by DE-miRNAs revealed that miRNAs may regulate genes involved in rubber biosynthesis. For instance, when Eu-miR45 expression declined, the expression of its predicted target, <i>small rubber particle protein 1</i> (<i>EuSRPP1</i>), increased in GZ511 vs. GB511 and GZ511 vs. GZ611, possibly resulting in higher rubber accumulation in GZ511. Additionally, we identified potential lncRNA−miRNA−mRNA networks in rubber biosynthesis. Overall, these results indicate that miRNAs play pivotal roles in regulating rubber biosynthesis via miRNA-target gene pairs and lncRNA−miRNA−mRNA networks in <i>E. ulmoides</i>. Our findings will enhance research on the mechanisms of rubber biosynthesis in plants.