Nanoplastics (NPs) have emerged as critical environmental contaminants, with growing concerns regarding their potential harm to organisms. Despite this, knowledge remains limited on whether NP exposure diminishes the capacity of organisms to respond to additional environmental stressors. In this study, we evaluated immune function in a burrowing tarantula, Chilobrachys guangxiensis, following NP exposure and subsequent challenges with lipopolysaccharide (LPS) and β-1,3-glucan. The total hemocyte count (THC) and hemolymph encapsulation rate were assessed to determine immune disruption. In addition, transcriptomic analyses were conducted to elucidate the mechanisms involved after both primary and secondary exposures. Results indicated that prolonged NP exposure did not cause significant changes in immunocompetence in C. guangxiensis. However, upon secondary exposure to LPS or β-1,3-glucan, individuals pre-exposed to NPs displayed significant changes in THC and impaired encapsulation capacity. Gene expression profiling based on quantitative real-time PCR revealed that LPS and β-1,3-glucan elicited varying immune responses and distinct gene expression profiles in NP-exposed C. guangxiensis. These findings suggest that NP exposure weakens immunocompetence in C. guangxiensis. This study provides comprehensive insights into the immune responses triggered by different pathogen-associated molecular patterns in NP-exposed C. guangxiensis, offering a novel perspective on the complex immunotoxicological effects of NP pollution.