Congenital syphilis is a vertically transmitted bacterial infection caused byTreponema pallidum, often causing multidomain neurodevelopmental disabilities. However, little is known about the pathogenesis of this disease. Brain organoids platform derived from the induced pluripotent stem cell (iPSC) is employed and exposed toT. palliduminfection for modelling congenital neurodevelopmental impairment. Single-cell RNA sequencing is used for identifying the subpopulations of differentially expressed genes and cellular heterogeneity and reconstructing differentiation trajectories followingT. palliduminfection. The results reveal thatT. palliduminfection influences the formation of neural rosette structures, reduces the cell number of the neural progenitor cell subcluster 1B (subNPC1B) and hindbrain neurons, and affects the neurodevelopment of the brain organoid. Moreover, it is speculated thatT. palliduminhibit the hindbrain neuron cell number through the suppression of subNPC1B subgroup in the organoids and inhibits transcription factor 3 activity in the subNPC1B-hindbrain neuronal axis. This is the first report on the inhibited effects ofT. pallidumon the neurodevelopment of the iPSC-derived brain organoid model. It is concluded thatT. pallidumcould inhibit the differentiation of subNPC1B in brain organoids, thereby reducing the differentiation from subNPC1B to hindbrain neurons, and ultimately affecting the development and maturation of hindbrain neurons.