B lymphocytes express multiple TLRs that regulate their cytokine production. We investigated the effect of TLR4 and TLR9 activation on receptor activator of NF-κB ligand (RANKL) expression by rat spleen B cells. Splenocytes or purified spleen B cells from Rowett rats were cultured with TLR4 ligand Escherichia coli LPS and/or TLR9 ligand CpG-oligodeoxynucleotide (CpG-ODN) for 2 days. RANKL mRNA expression and the percentage of RANKL-positive B cells were increased in rat splenocytes challenged by E. coli LPS alone. The increases were less pronounced when cells were treated with both CpG-ODN and E. coli LPS. Microarray analysis showed that expressions of multiple cyclin-dependent kinase (CDK) pathway-related genes were up-regulated only in cells treated with both E. coli LPS and CpG-ODN. This study suggests that CpG-ODN inhibits LPS-induced RANKL expression in rat B cells via regulation of the CDK pathway.