We investigated the effects of Ղ-glucan purified from Paenibacillus polymyxa JB115 on the viability and proliferation of splenocytes. Splenocytes play a critical role in host immunity. MTT assays and trypan blue exclusion tests revealed that Ղ-glucan significantly promoted the viability and proliferation of splenocytes over a range of concentrations. However, there was no specific subset change. Ղ-glucan protected splenocytes from cytokine withdrawal-induced spontaneous cell death. For further mechanistic studies, ELISA assay revealed that Ղ-glucan enhanced the expression of anti-apoptotic molecules and interleukin 7 (IL-7), a cytokine critical for lymphocyte survival. We also investigated the IL-2 dependency of Ղ-glucan-treated splenocytes to determine if treated cells could still undergo clonal expansion. In flow cytometric analysis, Ղ-glucan induced increased levels of the activation marker CD25 on the surface of splenocytes and Ղ-glucan-treated splenocytes showed higher proliferation rates in response to IL-2 treatment. This study demonstrates that Ղ-glucan can enhance the survival of splenocytes and provides valuable information to broaden the use of Ղ-glucan in research fields.