Abstract: In order to study the effect of FLASH radiotherapy (FLASH-RT) and conventional radiotherapy (CONV-RT) on gene expression profile in mouse spleen, and to provide theoretical basis for revealing the potential mechanisms of FLASH-RT, the mice experiments were carried out in different conditions. A total of 11 C57BL/6J male mice were divided into healthy control group (Ctrl group), CONV-RT group and FLASH-RT group according to random number table method. CONV-RT and FLASH-RT groups were irradiated in the abdomen of mice in the corresponding ways, with the dose of 12 Gy for both groups. And then the mice in 3 groups were killed by neck removal, spleen tissues were collected, and total RNA was extracted. Transcriptome sequencing (RNA-Seq) and bio-informatics analysis were used to investigate the changes of gene expression profiles in spleen tissue of mice after exposure. 936 differentially expressed genes (DEGs) are identified in group FLASH-RT vs CONV-RT. Among them, 321 genes are up-regulated and 615 genes are down-regulated. 1337 DEGs are identified in group FLASH-RT vs Ctrl. Among them, 322 genes are up-regulated and 1 015 genes are down-regulated. 1 082 DEGs are identified in groups of CONV-RT vs Ctrl. Among them, 445 genes are up-regulated and 637 genes are down-regulated. According to gene ontology (GO) analysis, these DEGs from group FLASH-RT vs CONV-RT involved various functions including defense response to other organism, other organisms cell, double-stranded RNA binding and so on. These DEGs from groups of FLASH-RT vs Ctrl involved various functions including defense response to other organisms, host cell cytoplasm, double-stranded RNA binding and so on. These DEGs from groups of FLASH-RT vs CONV-RT involved in several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including NOD-like receptor signaling pathway, antigen processing and presentation and so on. These DEGs from groups of FLASH-RT vs Ctrl involved in several KEGG pathways including Herpes simplex infection, NOD-like receptor signaling pathway and so on. Irf7 is likely to be activated by FLASH radiation. Gene expression profiles in mouse spleen tissues could be altered by FLASH radiation and CONV-RT radiation, and these DEGs involve multiple radiobiologically relevant functional pathways. FLASH-RT reduces radiation-induced spleen injury, and the mechanisms may be related to tissue immune response-induced radiation resistance.