Hazardous pollutants released into the real environment mostly own long-lasting cumulative characteristics and have progressively negative impacts on organisms, which are always neglected in laboratory toxicological tests. Here in this study, the different ecotoxicity of Ag nanoparticles (AgNPs) on earthworm Eisenia fetida was compared via various endpoints and transcriptional sequencing between the 28-day progressively repeated (from 60 to 80, final 100 mg/kg) and one-step (directly to 100 mg/kg) exposure. The results showed that earthworms under progressively repeated exposure showed significantly less biomass loss and reproductive inhibition, as well as lower Ag bioaccumulation (15.6 mg/kg) compared with one-step exposure (17.9 mg/kg). The increases in enzyme activities (superoxide enzyme and catalase) and gene expression (metallothionein) also implied higher antioxidant and genetic toxicity in one-step exposed earthworms compared with those from progressively repeated exposure. Furthermore, the transcriptomic analysis identified 582 and 854 differentially expressed genes in the treatments of one-step and repeated exposure respectively compared with the control group. The results of pathway annotation and classification suggested similar enrichments of damage induction but different in toxic stress responses, whereas earthworms from repeated exposure possessed more detoxification-related pathways like translation and multicellular organismal processes. This study innovatively took into account the impacts of processive exposure occurring in the real environment and elucidated distinctions of toxicity and adaptation caused by different exposure patterns, which provided the theoretical basis for real risk identification under the framework and guidance of traditional toxicology, also the implication for the improvement of eco-toxicological risk assessment.