Abstract Toxoplasma gondii is a ubiquitous pathogen that infects all warm‐blooded animals, including humans, causing substantial socioeconomic and healthcare burdens. However, there is no ideal vaccine for toxoplasmosis. As metabolism is important in the growth and virulence of Toxoplasma, some key pathways are promising antiparasitic targets. Here, we identified 6‐phosphogluconate dehydrogenase 1 (Tg6PGDH1) in the oxidative pentose phosphate pathway as a cytoplasmic protein that is dispensable for tachyzoite growth of T. gondii in vitro but critical for virulence and cyst formation in vivo. The depletion of Tg6PGDH1 causes decreased gene transcription involved in signal transduction, transcriptional regulation and virulence. Furthermore, we analysed the protective effect of the ME49Δ6pgdh1 mutant as an attenuated vaccine and found that ME49Δ6pgdh1 immunization stimulated strong protective immunity against lethal challenges and blocked cyst formation caused by reinfection. Furthermore, we showed that ME49Δ6pgdh1 immunization stimulated increased levels of interferon‐gamma, tumour necrosis factor‐alpha and Toxoplasma‐specific IgG antibodies. These data highlight the role of Tg6PGDH1 in the growth and virulence of T. gondii and its potential as a target for the development of a live‐attenuated vaccine.