The DPYSL5 (Dihydropyrimidinase like 5) gene encodes a cytosolic protein which regulates axonal guidance and neuritic growth. We initially described two missense mutations (p.Glu41Lys, recurrent ; p.Gly47Arg) in DPYSL5 gene in nine individuals presenting intellectual disability and agenesis of the corpus callosum. Interestingly, both variants caused abnormal neuronal development and impaired interaction between DPYSL5 and MAP2 and TUBB3 proteins. Recently, a third de novo variant (p.Glu25Lys) was identified in four individuals with a similar clinical phenotype. Various studies described DPYSL5 protein expression in synaptic fractions, suggesting a role in the mature neuron. Our aim is to clarify the physiological and pathological role of DPYSL5 at the synaptic level. We studied primary neuronal cultures transfected with wildtype (WT) or each of the DPYSL5 variants to evaluate their impact on synapse development and maturation. The overexpression of the WT-DPYSL5 form increased synaptic density and dendritic spine maturation. In contrast, the p.Glu25Lys variant does not induce an increase in the total number of synapses, and the p.Glu41Lys and p.Gly47Arg variants result in a loss of effect of DPYSL5 on dendritic spine maturation, suggesting a loss-of-function impact. In brain homogenates from Dpysl5 -/- mouse models, we found that the expression of PSD95 and vGLUT1 proteins was impaired compared to WT mice. Reduced expression level of synaptic proteins was also revealed in brain extracts from our Dpysl5WT/p.Glu41Lys mouse model (i.e carrying the recurrent variant). These results suggest the involvement of DPYSL5 in synaptogenesis and synaptic transmission, and further confirm its physiopathological contribution in neurodevelopmental disorders.