The earthworm Eisenia fetida is frequently used in ecotoxicological studies; however, it has not yet been investigated using proton nuclear magnetic resonance (1H NMR) metabolic profiling methods. The present study investigates the impact of depuration time, sample homogenization, and different extraction solvents on the quality and reproducibility of the 1H NMR spectra of E. fetida with the goal of determining whether this species is suitable for future metabonomic studies. A depuration time of 96 h, followed by intact lyophilization before homogenization and extraction into a deuterium oxide (D2O)-based phosphate buffer, was found to produce extracts with excellent 1H NMR reproducibility. The D2O buffer extracted the largest quantity of the widest variety of earthworm metabolites, which is consistent with the results from other studies using different earthworm species. Nuclear magnetic resonance assignments of the major metabolites in the D2O-based buffer also were performed and found to be similar to those for other earthworm species, such as Eisenia veneta, but also to have characteristic attributes in E. fetida. The major metabolites identified include amino acids (alanine, arginine, glutamic acid, glutamine, glycine, leucine, lysine, phenylalanine, serine, tyrosine, and valine), two sugars (glucose and maltose), the sugar alcohol mannitol, and the polyalcohol inositol. Two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) also were examined using protocols developed for E. fetida, and of the three species, the 1H NMR spectra of E. fetida had the least variation, indicating this species is well-suited for future metabolomic-based ecotoxicity studies.