Glucocorticoid hormones have been widely used in clinical practice as potent anti-inflammatory and immunosuppressive agents. However, the underlying mechanisms of how they work remain unaddressed. Here, we used RNA-set to profile spleen gene expression in adult mice after consistent intraperitoneal injection of dexamethasone. We identified 13565 genes in control (injected with 0.9% NaCl) and 13702 genes in dexamethasone-injected group, 12920 genes were expressed in both, but 645 genes were identified only in control and 782 genes were identified only in the dexamethasone group. In the dexamethasone-injected group 101 and 67 genes were down-and up-regulated, respectively. Among these, 129 were coding genes, 19 were identified as non-coding genes or pseudogenes, and the remaining 20 were TEC (to be experimentally confirmed) genes. Gene ontology (GO) and KEGG pathway analysis revealed that the cytokine-cytokine receptor interaction pathway was highly enriched in these differentially expressed genes, and that a majority of the 129 identified coding genes were involved in immune system and cell adhesion-associated processes. Moreover, systemic lupus erythematosus, renin-angiotensin system, fat digestion and absorption, and glycerolipid metabolism pathways were significantly affected in the dexamethasone-treatment group. No obvious signaling pathway was enriched in the control group. Additionally, 20 immunoglobulin heavy or light chain variable region genes (IGH(L)Vs) were down regulated in the dexamethasone-injected group. IGH(L)Vs encode the variable region of immunoglobulin heavy chain and determine the diversity and specificity of antibodies. We were unable to determine the function of the 19 non-coding genes with differential expression following dexamethasone treatment. Our findings indicate that the expression of IGH(L)Vs and non-coding genes play an important role in the anti-inflammatory and immunosuppressive effects of dexamethasone and could be developed as potential agents in clinical practice.