Nanoparticles (NPs), having exceptional physicochemical and electrical characteristics with lower toxicity, have evolved as dynamic drug delivery carriers in living organisms. Potentially, the intragastric gavage of silica nanoparticles (SiNPs) affects gut microbiota profiles in immunodeficient mice. In this study, the impact of SiNPs of variable size and dosage was investigated in cyclophosphamide (Cy)-induced immunodeficient mice, specifically on their immune functions and gut microbiota, through physicochemical and metagenomic analysis. SiNPs of different sizes and doses were gavaged to Cy-induced immunodeficient mice for 12 days at an interval of 24 h to investigate their effects on immunological functions and the gut microbiome of mice. Our results showed that SiNPs had no significant toxicological effects on the cellular and hematological activities of immunodeficient mice. Furthermore, after the administration of different levels of SiNPs, no immune dysfunction was found in the immunosuppressed mice groups. However, gut-microbial studies and comparisons of characteristic bacterial diversity and compositions demonstrated that SiNPs significantly affect the abundance of different bacterial communities. LEfSe analysis revealed that SiNPs significantly increased the abundance ofLactobacillus,Sphingomonas,Sutterella,Akkermansia, andPrevotella, and potentially reducedRuminococcusandAllobaculum. Thus, SiNPs significantly regulate and modify the configuration of the gut microbiota in immunodeficient mice. These dynamic variations in the intestinal bacterial community, abundance, and diversity provide new insight into the regulation and administration of silica-based NPs. This would be helpful for the further demonstration of the mechanism of action and prediction of the potential effects of SiNPs.