Kinesin 1 (KIF5) is one of the major types of motor proteins that transport synaptic proteins and mRNAs to dendrites, but their function in dendritic transport of neurons in vivo remains undefined. Using in vivo two-photon longitudinal imaging, we found that mice with conditional knockout of kif5b (KIF5B cKO) in CaMKIIα-Cre expressing neurons showed heightened turnover and lower stability of dendritic spines in L2/3 pyramidal neurons in the frontal association cortex. The increase of dendritic spine instability was accompanied with less postsynaptic density 95 (PSD95) acquisition in dendritic spines without affecting the inhibitory synaptic scaffolding protein gephyrin. Notably, the RNA-binding protein Fragile X Mental Retardation protein (FMRP), a KIF5 cargo that regulates PSD95 translation and degradation, was translocated to the proximity of newly formed spines several hours before the spine formation events in vivo in control mice. However, this preceding transport of FMRP towards new spines was abolished in KIF5B cKO and the FMRP puncta became less dynamic in dendritic spines. We further found that FMRP was localized closer to newly formed spines after fear extinction, and this learning-dependent localization of FMRP was disrupted in KIF5B cKO. Together, these results indicate that KIF5B is a crucial motor protein for the dendritic targeting of synaptic proteins that underlies dendritic spine plasticity.