Long-term memory formation requires de novo RNA and protein synthesis. By using the differential display-polymerase chain reaction strategy, we have presently identified the Nedd4 family interacting protein 1 (Ndfip1) cDNA fragment that is differentially expressed between the slow learners and the fast learners from the water maze learning task in rats. Further, the fast learners show decreased Ndfip1 mRNA and protein expression levels than the slow learners. Spatial training similarly decreases the Ndfip1 mRNA and protein expression levels. Conversely, the Ndfip1 conditional heterozygous (cHet) mice show enhanced spatial memory performance compared to the Ndfip1flox/WT control mice. Result from co-immunoprecipitation experiment indicates that spatial training decreases the association between Ndfip1 and the E3 ubiquitin ligase Nedd4 (Nedd4-1), and we have shown that both Beclin 1 and PTEN are endogenous ubiquitination targets of Nedd4 in the hippocampus. Further, spatial training decreases endogenous Beclin 1 and PTEN ubiquitination, and increases Beclin 1 and PTEN expression in the hippocampus. On the other hand, the Becn1 conditional knockout (cKO) mice and the Pten cKO mice both show impaired spatial learning and memory performance. Moreover, the expression level of Beclin 1 and PTEN is higher in the Ndfip1 cHet mice compared with the Ndfip1flox/WT control mice. Here, we have identified Ndfip1 as a candidate novel negative regulation for spatial memory formation and this is associated with increased ubiquitination of Beclin 1 and PTEN in the hippocampus.