Human plasma metabolomics dataset generated from controlled human hookworm infection study. Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed to prevent or treat various intestinal and extraintestinal disease. However, full-scale clinical trials examining hookworm therapy are limited by the inability to scale-up the production of hookworm larvae to infect sufficient numbers of patients. With the aim of overcoming this challenge, this study infected four healthy individuals with hookworm larvae that had been reanimated from cryopreserved eggs, to examine their viability and immunogenicity. We demonstrate that reanimated cryopreserved hookworm larvae establish a viable hookworm infection and elicit a similar immune response to larvae cultured from fresh stool. Furthermore, a refined understanding of the therapeutic mechanisms of hookworm is imperative to determine which diseases to target with hookworm therapy. To investigate potential therapeutic mechanisms, this study infected 13 healthy individuals with hookworm and assessed changes in the immune cells, microbiome, and metabolome. We identified potential immunoregulatory mechanisms by which hookworm may provide a beneficial effect on its host, including increased expression of CTLA-4 on regulatory T cells (Tregs) and upregulation of tryptophan metabolism. In summary, our findings demonstrate the feasibility of full-scale clinical trials examining hookworm therapy by minimising the reliance on human donors and optimising the culturing process, thereby enabling viable hookworm larvae to be mass-produced and enabling on-demand inoculation of patients. Furthermore, this study provides insights into the complex interactions between helminths and their host, which could inform the development of novel therapeutic strategies.