Abstract: Nano-zero-valent iron (nZVI) is widely used to remedy soil heavy metal pollution. However, the potential effects of nZVI on soil invertebrates, soil quality and microbial communities have not been well studied. In this study, we used Eisenia foetida (0, 10 pieces per kilogram soil) as the test species and examined the potential effects of nZVI (mass ratios of 0, 0.05%, 0.25%, and 0.50%) on the earthworm-bacteria-soil ecosystems after 15, 30, and 45 days of exposure. The results showed that after 45 days of exposure, there was no significant difference in survival rate and biomass of earthworms. The earthworm survival rate and content of malondialdehyde in the 0.50% nZVI system decreased by 27.66% and 0.86 nmol∙g⁻¹, respectively, compared with those on day 15. However, the earthworm biomass increased by 1.20 times, and the catalase activity increased by 2.62 times. At the phylum or genus level, nZVI had no significant effects on the relative abundance, diversity index, and abundance index of soil microorganisms. Compared with the 0 nZVI system, the proportion of soil large aggregates (>250 μm), the average weight diameter of soil aggregates, and the content of available phosphorus (P) in the 0.50% nZVI system increased by 15.69%, 12.59%, and 21.20% under earthworm-mediated conditions, respectively. The proportion of soil macroaggregates and the average weight diameter of soil aggregates in the earthworm and nZVI composite systems were significantly higher than those in the corresponding single nZVI system, and earthworm activity significantly improved the stability of soil aggregates under nZVI stress (P<0.01). In this study, we found that long-term exposure to nZVI had no significant toxic effects on the community characteristics of soil microorganisms but promoted the growth of earthworms, which further improved the bioavailability of soil nutrients. This study provides a scientific basis for environmental safety assessments of nZVI in soil restoration applications.