Withania somnifera(L.) Dunal (familySolanaceae) is a medicinal plant known for, among many pharmacological properties, an immune boosting effect. Our recent study revealed that its key immunostimulatory factor is lipopolysaccharide of plant-associated bacteria. This is peculiar, because, although LPS can elicit protective immunity, it is an extremely potent pro-inflammatory toxin (endotoxin). However,W. somniferais not associated with such toxicity. In fact, despite the presence of LPS, it does not trigger massive inflammatory responses in macrophages. To gain insights into the safe immunostimulatory effect ofW. somnifera, we conducted a mechanistic study on its major phytochemical constituent, withaferin A, which is known for anti-inflammatory activity. Endotoxin-triggered immunological responses in the presence and absence of withaferin A were characterized by both in vitro macrophage-based assay and in vivo cytokine profiling in mice. Collectively, our results demonstrate that withaferin A selectively attenuates the pro-inflammatory signaling triggered by endotoxin without impairing other immunological pathways. This finding provides a new conceptual framework to understand the safe immune-boosting effect ofW. somniferaand possibly other medicinal plants. Furthermore, the finding opens a new opportunity to facilitate the development of safe immunotherapeutic agents, such as vaccine adjuvants.