Immune response is a necessary self-defense mechanism that protects the host from infectious organisms. Many medicinal plants are popularly used in Asian folk medicine to increase body resistance. An herbal formulation named KM1608 was prepared from three medicinal plants: , , and . In this study, we evaluated the immune stimulatory effect of KM1608 on RAW 264.7 murine macrophages. Network pharmacological analyses were used to predict potential immune response pathways of major compounds from KM1608. The cytotoxicity and immuno-stimulating effect of KM1608 were determined using cell viability and nitric oxide assays. The underlying mechanism of immunomodulatory activity was evaluated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) of pro-inflammatory cytokines. The results of network pharmacological analysis suggested that major compounds from KM1608 possess anticancer potential via immune signaling pathways. After treatment with KM1608 at 25-100 µg/mL for 24 h, the level of nitric oxide was increased in the dose-dependent manner. The results of quantitative real-time PCR showed that KM1608 stimulates the expression of immune cytokines (interferon (IFN)-α, -β, IL-1β, -6, IL-10, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2)) in macrophages. KM1608 extract is a potential agent for immune response enhancement.