Protaetia brevitarsis seulensis (P. brevitarsis) larvae traditionally has been used as alternative medicine. Although various health benefits have been reported, immunomodulatory effects of P. brevitarsis extract are still unknown. In this study, the immuno-enhancing activity of P. brevitarsis larvae hot-water extract (PWE) was investigated using RAW264.7 macrophage cell line. PWE did not exert cytotoxicity at concentrations ranging from 1 to 200 μg/mL in RAW264.7 cells. The treatment of PWE increased the production of pro-inflammatory cytokines (tumor necrosis factor-alpha; TNF-α and interleukin-6; IL-6) at dose ranging from 100, 200 μg/mL in RAW264.7 cells. PWE increased inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production in RAW264.7 cell. Additionally, treatment of PWE (100, 200 μg/mL) induced expression of surface molecules (cluster of differentiation; CD80/86 and major histocompatibility complex; MHC-class Ⅰ/II) in RAW264.7 cells. These immunomodulatory effects of PWE was mediated by mitogen-activated protein kinases (serine/threonine protein kinase; p38, c-Jun N-terminal kinase; JNK, extracellular signal-regulated kinase; ERK) phosphorylation and nuclear factor-κB translocation. In order to examine its potential as a chemotherapy supplement, we investigated whether PWE protects cisplatin-induced cell death in RAW264.7 cells. Pre-treatment of PWE (100, 200 μg/mL) alleviated cisplatin-induced cytotoxicity, early- and late- apoptosis population. Furthermore, pretreatment of PWE (100, 200 μg/mL) reduced cisplatin-induced reactive oxygen species (ROS) production. These PWE-induced cytoprotective actions was mediated by inhibition of Bcl-2-associated X protein (Bax) and cleaved-caspase 3. In conclusion, these findings provide experimental evidence that PWE can be utilized as an nutraceutical ingredient for chemotherapy-induced cytotoxicity protection and immune-enhancing.