Selenium (Se) is an essential trace element and its deficiency can lead to immune dysfunction. Many studies have investigated the immune damage caused by Se deficiency in chickens, but its mechanism still needs to be explored. In this study, we fed 1-day-old Hyline male chickens with Se deficient diets (the Se content was 0.008 mg kg-1 of diet) and a basal diet (the Se content was 0.15 mg kg-1 of diet). The spleen was collected at the sixth week and used for subsequent experiments. The pathological analysis showed that Se deficiency leads to the destruction of the normal nuclear structure of the spleen cell, and we can observe obvious chromatin condensation and nuclear debris. We constructed a transcriptome database and analyzed the abundance of various genes in the spleen by transcriptome sequence. The analysis of differentially expressed genes (DEGS) showed significant changes in 337 genes, including 210 up-regulations and 127 down-regulations after feeding Se deficient diets. Se deficiency can significantly change oxidative stress and inflammatory response genes in chicken spleen. This study confirmed that Se deficiency increased the IL-2 levels, whereas it down-regulated IL-17, IFN-γ and Foxp3, which indicates that the immune dysfunction of the spleen and Th1/Th2 is imbalanced. We also found that Se deficiency down-regulated some related genes for endoplasmic reticulum Ca2+ transport, leading to endoplasmic reticulum stress (ERS). Moreover, we determined that Se deficiency triggered the low expression of DUSP1/NF-κB. In summary, our results indicate that Se deficiency can inhibit the spleen immune function of chickens by regulating the DUSP1/NF-κB pathway and ERS, leading to spleen damage in chickens. Based on transcriptomics research, our results will help further study the harmful effects of Se deficiency.