Methionine aminopeptidases (MAPs), which remove the N-terminal methionine from newly synthesised proteins, are present in all life forms. Three type I MAPs and one type II MAP are encoded in the genome of Toxoplasma gondii. In this study, we found that the inducible knockdown of each type I TgMAP (TgMAP1a–c) reduced the growth and proliferation of the parasite significantly. Among them, TgMAP1c was found to be localised to the apicoplast of the parasite. Inducible knockdown of TgMAP1c led to a defect in the abundance of apicoplast-encoded transcripts, and a later reduction in the apicoplast genome copy number and loss of the apicoplast structure. This finding indicates that transcription of the apicoplast genome was impaired upon knockdown of TgMAP1c. We also found that the function of TgMAP1c in apicoplast biogenesis depends on its enzymatic domain. Expression of a recombinant protein in which the active domain of TgMAP1c was replaced with that of TgMAP1a or TgMAP1b could not restore the defective growth and replication phenotype caused by knockdown of TgMAP1c, indicating that these three enzymes have distinct substrate preferences. An in vitro analysis also revealed that TgMAP1c is an active enzyme that acts specifically on the substrate H-Met-p-NA. In addition, inducible knockdown of TgMAP1c reduced the virulence of T. gondii in mice. Taken together, these results demonstrate that TgMAP1c plays a key role in the biogenesis and maintenance of the T. gondii apicoplast.