The Bursa of Fabricius (BF), a central lymphoid organ in birds, is of importance to B cell differentiation and immune modulation. Bursopentin (BP5) is an immune regulatory bioactive peptide derived from BF. In this study, the proliferation and transcriptome profiles of chicken pre-B DT40 cells treated with or without BP5 were investigated by cell proliferation assay and microarray analysis. The results indicated that BP5 showed concentration dependent dual-effects on the proliferation of DT40 cells. According to the results of gene microarray, 3022 genes were differentially expressed. The expression levels of randomly selected differential genes were verified by real time PCR, which were correlated with the microarray results. And the results of GO analysis showed that the differential expression genes were mainly involved in biological processes of innate immune response. Also, KEGG analysis indicates that toll like receptor (TLR) and NF-kappa B signaling pathway may play important roles in DT40 cells treated with BP5. These results will provide a valuable basis for understanding the potential molecular mechanism of BP5 on B lymphocyte.