Objective By observing the effect of subarachnoid anesthesia of fentanyl on the transcriptomic expression of spinal dorsal root ganglion (DRG) in rats, the possible anesthesia mechanism was investigated. Methods Six male SD rats aged 5 to 6 weeks were selected and divided into normal saline group (group N) and fentanyl group (group F) by random number table method, with 3 rats in each group.After injection of 20ul of normal saline or equal volume of fentanyl into the subarachnoid space, extract the dorsal root ganglion of the spinal cord for mRNA sequencing (RNAseq) and screen for differentially expressed genes.According to the sequencing results, miRNAs were screened and cluster analysis was performed. On this basis, key miRNAs with subarachnoid injection of fentanyl on DRG were screened through Wayne diagram, and target genes were predicted by gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, etc. Results After determining the quality of the samples, high-throughput sequencing detected that 58 genes were significantly up-regulated and 54 genes were significantly down-regulated in spinal dorsal root ganglia of rats after subarachnoid anesthesia of fentanyl.By GO and KEGG analysis, it was found that the pathway with significant changes during subarachnoid anesthesia of fentanyl was mainly related to ion transmembrane transport, calcium channel release activity, SNARE(related to CGRP secretion) in vesicle transport. Conclusion After subarachnoid anesthesia with fentanyl, the expression of miRNA in dorsal root ganglia of spinal cord was significantly changed.Analysis of relevant functional changes suggested that related mirNA-targeting genes such as ion transmembrane transport, calcium channel release activity, SNARE(related to CGRP secretion) in vesicle transport may be involved in the anesthesia regulation of fentanyl subarachnoid space.