Contamination of roxarsone has been recognized as a potential environmental hazard. In this study, Eisenia fetida samples were collected after roxarsone exposures to analyze their intestinal epithelium ultrastructure, expression levels of stress-related genes, and proteomics. Our results showed that mitochondria and endoplasmic reticulum in roxarsone-treated earthworms demonstrated variety of damages. Furthermore, 149 proteins were displayed in 2-DE, and 36 of them were identified by MALDI-TOF/TOF-MS. Those identified proteins are involved in several important processes including cell immunity, cell stress responses, and cell genetic behaviors. Our study demonstrates the toxicity responses of earthworms toward arsenic-based animal drug roxarsone with practical usefulness and demonstrates a proteomic profile change that may be critical for the roxarsone stress survival mechanisms of E. fetida. Graphical Abstract Inspiration of this referred to the form of Fig. 4 in the article "Proteomic analysis of a high aluminum tolerant yeast Rhodotorula taiwanensis RS1 in response to aluminum stress" of Chao, W et al.