The entomopathogenic fungus (EPF) Ophiocordyceps sinensis has a long-term coexistence with its host insect, Thitarodes xiaojinensis, making it a unique model for host–pathogen interactions. Hemolymph, a critical component in insects, plays an essential role in maintaining both nutritional and immune homeostasis. However, the mechanism of the host’s immune response remains unclear when O. sinensis proliferates in the hemolymph. O. sinensis caused damage to the insect’s intestinal barrier, facilitating the translocation of gut bacteria into the hemocoel. Subsequently, the presence of O. sinensis and opportunistic pathogenic bacteria from the gut disrupted the homeostasis of the hemolymph microbiota, resulting in an increase in bacterial diversity. This disruption triggered a series of physiological responses in the host, including elevated levels of endocrine hormones specifically 20-hydroxyecdysone (20E) and juvenile hormone 3 (JH3). Additionally, there was an enhancement of antioxidant capacity, as indicated by increased total antioxidant capacity and glutathione S-transferase activity, along with the production of antimicrobial peptides (AMPs) as part of the immune defense. Notably, the rise in 20E levels during O. sinensis infection might have significantly contributed to the increased production of AMPs. O. sinensis infection significantly alters T. xiaojinensis physiology. Humoral immunity in infected hosts is primarily in response to hemolymph microbial homeostasis due to intestinal translocation. Among them, 20E upregulates AMP-related genes, suggesting a key immune strategy for managing microbial imbalances while tolerating fungal pathogens.