AbstractSynaptic modification in postnatal development is essential for the maturation of neural networks. Developmental maturation of excitatory synapses occurs at the loci of dendritic spines that are dynamically regulated by growth and pruning. Striatal spiny projection neurons (SPNs) receive excitatory input from the cerebral cortex and thalamus. SPNs of the striatonigral direct pathway (dSPNs) and SPNs of the striatopallidal indirect pathway (iSPNs) have different developmental roots and functions. The spatial and temporal dynamics of dendritic spine maturation of these two types of SPNs remain elusive. Here, we delineate the developmental trajectories of dendritic spines of dSPNs and iSPNs in the caudoputamen and nucleus accumbens (NAc). We labeled dendritic spines of SPNs by microinjecting Cre-dependent AAV-eYFP viruses into newborn Drd1-Cre or Adora2a-Cre mice, and analyzed spinogenesis at three levels, including different SPN cell types, subregions and postnatal times. In the dorsolateral striatum, spine pruning of dSPNs and iSPNs occurred at postnatal day (P)30–P50. In the dorsomedial striatum, the spine density of both dSPNs and iSPNs reached its peak between P30 and P50, and spine pruning occurred after P30 and P50, respectively, for dSPNs and iSPNs. In the NAc shell, spines of dSPNs and iSPNs were pruned after P21–P30, but no significant pruning was observed in iSPNs of lateral NAc shell. In the NAc core, the spine density of dSPNs and iSPNs reached its peak at P21 and P30, respectively, and subsequently declined. Collectively, the developmental maturation of dendritic spines in dSPNs and iSPNs follows distinct spatiotemporal trajectories in the dorsal and ventral striatum.