Objective To conduct sequencing and bioinformatics analysis of mRNA from mouse macrophages regulated by Mn^2＋, screen out differentially expressed genes, and analyze their biological functions, so as to explore the regulatory effect of Mn^2＋ on mouse macrophages. Methods MnCl₂ and NaCl were used to treat mouse peritoneal macrophages for 24 h, respectively. The mRNA-seq method was used to screen for the differentially expressed genes of macrophages regulated by Mn^2＋, then the differentially expressed genes were analyzed with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and the key differentially expressed genes were verified by quantitative polymerase chain reaction (qPCR). Results A total of 1 637 differentially expressed genes were obtained by integrating database information and analyses, including 912 upregulated genes and 725 down-regulated genes. GO enrichment analysis showed that these differentially expressed genes had biological activities, such as phagocytosis, cell proliferation and differentiation, and regulation of transcription factors. The KEGG pathway was mainly enriched in phosphatidylinositol 3-kinase-protein kinase B and nuclear factor κB signaling pathways. The top 10 genes associated with immunity and with the most significant differences were Fc receptor, immunoglobulin G, high affinity Ⅰ (Fcgr1), sterile α motif domain containing 11 (Samd11), activity regulated cytoskeleton-associated protein (Arc), G protein-coupled receptor 35 (Gpr35), small proline-rich protein 2H (Sprr2h), Wnt family member 4 (Wnt4), integral membrane protein 2A (Itm2a), atonal bHLH transcription factor 8 (Atoh8), ubiquitin conjugating enzyme E2C (Ube2c) and small proline rich protein 2B (Sprr2b). The qPCR verification results of the 10 genes were consistent with the trend of mRNA sequencing analysis. Conclusion The screening of the differentially expressed genes from mouse macrophages regulated by Mn^2＋ based on mRNA-seq data and the analyses of GO and KEGG enrichment show that Mn^2＋ has an immunoregulation effect on mouse macrophages.