L-Theanine is a nonprotein amino acid in tea, and its immunomodulatory function has been confirmed. This study aimed to investigate the effect of L-theanine addition on cytokines secretion in rat splenic lymphocytes and explore its potential immunomodulatory effects on the mevalonate biosynthetic pathway. Our results showed that L-theanine treatment did not influence the proliferation and division indexes of the splenic lymphocytes subsets. Interestingly, L-theanine treatment had regulated the contents of IFN-γ, IL-2, IL-4, IL-10, IL-12, and TNF-α (P < 0.001) except IL-6 and upregulated the mRNA and protein expression of Ras-related protein Rap-1A (Rap1A), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and farnesyl diphosphate synthase (FDPs) (P < 0.001). Additionally, there was a positive correlation between Rap1A and HMGCR proteins expression and IFN-γ, IL-4, and IL-6 levels. In conclusion, L-theanine regulated the secretion of cytokines probably by activating expression of Rap1A and HMGCR proteins involved in the mevalonate biosynthetic pathway in rat splenic lymphocytes. Therefore, L-theanine might be a promising potential drug candidate as immunopotentiator.