Toxoplasma gondii is a widespread protozoan with a complex life cycle, characterized by transitions between various hosts and developmental stages, each tailored to a specific niche within its host. However, the regulatory mechanisms governing these life cycle transitions are not well understood. In this study, we investigated the AP2 factor AP2X-1, which is expressed during the tachyzoite and bradyzoite stages but decreases in the mature merozoite stage. Knockout of ap2X-1 significantly impaired tachyzoite invasion and replication while increasing the frequency of bradyzoite differentiation. As a component associated with the HDAC3/MORC complex, loss of ap2X-1 led to the upregulation of bradyzoite- and sexual stage-specific genes. Single-cell sequencing revealed that ap2X-1 knockout strain exhibited a mixed population of tachyzoite-, bradyzoite-, merozoite-, and sporozoite-like parasites. CUT&Tag analysis revealed a substantial overlap between AP2X-1 and HDAC3/MORC complex at the promoters of bradyzoite- and sexual stage-specific genes. Additionally, ATAC-seq analysis demonstrated that AP2X-1 influences chromatin compaction and accessibility, suggesting that AP2X-1 may modulate the function of the HDAC3/MORC complex to facilitate the repression of bradyzoite differentiation and sexual commitment. Loss of ap2X-1 resulted in a significant attenuation of T. gondii virulence and decreased brain cyst formation in vivo. These findings identify AP2X-1 as a critical negative regulator of T. gondii sexual development.