Calcium/Calmodulin-dependent kinase II (CaMKII) is a serine/threonine kinase with a wide range of substrates. A number of studies have established that CaMKII is fundamentally important for various learning and memory processes. Given this importance the activity of CaMKII must be tightly regulated. Two endogenous inhibitor proteins of CaMKII, CaMK2N1 and CaMK2N2, have been identified. During contextual fear memory formation CaMK2N1 and CaMK2N2 expression increases in brain regions that are related to the task. However, the functions of CaMK2Ns are still unknown. Our aim was to study the physiological roles of these inhibitors in memory and learning. For that purpose we used adeno-associated virus vector to either knockdown the expression of CaMK2N1 or overexpress CaMK2N2 in the dorsal hippocampus of mice. Animals were trained and tested in contextual fear conditioning paradigm. The knockdown of CaMK2N1 expression had no effect on long-term memory formation, but it impaired long-term memory maintenance after retrieval. Western blot analyses revealed that CaMK2N1 knockdown prevents a decrease in T286 phosphorylation of αCaMKII induced by memory testing, as well as a reduction of GluA1 and c-fos levels. This puts forward the hypothesis that CaMK2N1 is necessary for inducing a decrease in neuronal activation after memory retrieval and that this process is required for memory maintenance. Regarding the hippocampal overexpression of CaMK2N2, treatment prior to training blocked contextual memory formation. On the other hand, overexpression of the same inhibitor after training had no effect on contextual long-term memory maintenance. These results support the view persistent CaMKII activity is not the molecular basis of long-term memory maintenance but its inhibition is.