Background <p><i>Toxoplasma gondii</i> is an opportunistic intracellular parasite that can cause severe reproductive disorders during pregnancy. Progesterone is markedly elevated during pregnancy and has been shown to affect the replication of T. gondii. However, the downstream cellular processes and molecular mechanisms underlying progesterone-mediated regulation of parasite replication remain unclear.</p> Methods <p>Transcriptomic analysis was performed to investigate the global gene expression changes in tachyzoites after progesterone treatment. Differentially expressed genes were subjected to functional enrichment analysis. The effects of progesterone on parasite division were further assessed by immunofluorescence assays targeting subpellicular microtubules, centrosome, and organelles.</p> Results <p>Transcriptomic analysis identified 329 differentially expressed genes after progesterone treatment, which were mainly enriched in microtubule-associated pathways, including microtubule motor activity and microtubule-based movement. Although the overall structure of subpellicular microtubules showed no detectable alteration, progesterone selectively disrupted division of the outer core of the centrosome, while the inner core of the centrosome was largely unaffected. Further analysis of organelle division showed that progesterone mainly interfered with early events of endodyogeny, including the segregation of the centrosome, Golgi, and apicoplast, whereas later division-related structures were less affected.</p> Conclusions <p>These findings indicate that progesterone impairs T. gondii replication by selectively interfering with microtubule-dependent processes, especially outer core centrosome division. This study provides new insight into how the pregnancy-associated hormone progesterone regulates parasite replication.</p> Graphical Abstract <p></p>