Background: Periplaneta americana L . is one of the most famous traditional Chinese medicines (TCMs) , and CII-3 is the major bioactive extract of Periplaneta americana L. In recent years, CII-3 has gradually attracted the attention of researchers for its powerful capacity for treating immunocompromised diseases. However, systematical chemical composition investigation and mechanisms on immunomodulation of CII-3 have not been thoroughly scrutinized. Methods: The chemical ingredients of CII-3 were determined by ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), and immune-enhancing mechanisms and active constituents were investigated by integration of metabolomics with network pharmacology and molecular docking. Results: A total of 25 components were identified in the aqueous extract of CII-3, among which 10 were unambiguously confirmed by comparison with reference standards. In immunosuppressed rats, oral administration of CII-3 normalized biochemical profiles by promoting the secretion of immune-related cytokines (IL-2, IL-6) and immunoglobulins (IgG, IgM), significantly improving immune organ indices, and alleviating pathological lesions in the thymus and spleen. Furthermore, CII-3 stimulation attenuated CTX-induced upregulation of IL-6 mRNA levels and concurrently enhanced IL-2 mRNA expression in these two immune organs. Based on metabolomics analysis, 27 differential metabolites along with 7 crucial metabolic pathways including starch and sucrose metabolism, arginine biosynthesis, porphyrin and chlorophyll metabolism, nicotinate and nicotinamide metabolism, steroid biosynthesis, pyrimidine metabolism and tryptophan metabolism were regulated after CII-3 treatment, suggesting that CII-3 has the potential to target these metabolites and key pathways to improve immunosuppression. Nitric oxide synthase 1 (NOS1), nitric oxide synthase 3 (NOS3), acetylcholinesterase (ACHE), cluster of differentiation 38 (CD38) and poly (ADP-ribose) polymerase 1 (PARP1) were considered as the pivotal targets, and ginsenine, cyclo (Tyr-Asp), guanosine, tryptophan and inosine were deemed potential active components of CII-3 in alleviating immunosuppression. Conclusion: The proposed approach provides a valuable evidence for demonstrating the material basis of CII-3. The results obtained in the present study elucidated that the potential immunomodulatory activity of CII-3 might be closely associated with these crucial targets, which can modulate the levels of major metabolites through multiple metabolic pathways, thus ameliorating immunosuppression.