Recent studies have reported on the molecular mechanisms underlying dendritic spine (spine) dynamics. Because most of these studies investigated spine dynamics by overexpressing constitutively active or dominant-negative PSD (postsynaptic density) proteins in cultured mature neurons, the results represent the enlargement of mature spines or their return to an immature state. Here, we developed the technique of in utero electroporation to investigate spine dynamics. Using this technique, we demonstrated the suppression of spine maturation by the C-terminal variants of PSD-Zip70 in vitro and in vivo. Transient overexpression of the C terminus of PSD-Zip70 and knock-down of PSD-Zip70 also displayed the destabilization of mature spines. We further found the PSD-Zip70 and SPAR (spine-associated RapGAP) interaction via the short C-terminal region of PSD-Zip70 and the GK-binding domain of SPAR. In association with immature spines induced by overexpression of the PSD-Zip70 C terminus or knock-down of PSD-Zip70, SPAR lost its spine localization. Overexpression of the GK-binding domain of SPAR also induced to form immature spines without affecting the localization of PSD-Zip70 in the small heads of filopodial spines. Our results suggest that PSD-Zip70 in collaboration with SPAR is critically involved in spine maturity, especially in the mature spine formation and the maintenance of spine maturity.