In recent years, animals and plants have received increasing attention as potential next-generation protein production systems, especially for biopharmaceuticals and animal proteins. The aim of the present study was to develop the earthworms Eisenia fetida Waki and Eisenia andrei Sagami as next-generation animal protein production hosts. These earthworms have been approved as model animals for acute toxicity tests by the Organization for Economic Co-operation and Development, and they have post-translational modification systems. However, so far, none of the studies have used earthworm transfection techniques. Thus, we developed a transfection method for E. fetida and E. andrei using microinjection and electroporation systems. The maximum survival rates and transfection efficiencies were 79.2% and 29.2% for E. fetida, and 95.8% and 50.0% for E. andrei, respectively. Furthermore, human erythropoietin was detected in the transformed earthworm tail fragments using an enzyme-linked immunosorbent assay. These results contribute to the development of a potential earthworm-based novel animal protein production system.