Nucleic acid fraction (NAF) of hydrolyzed salmon milt extract has been reported to enhance both object recognition and location memories in healthy mice and promote proliferation of primary cultured neural stem cells (pcNSCs). NAF highly contains tri-deoxyribonucleotides, and only tri-deoxycytidine (CCC) among all 64 tri-deoxyribonucleotides significantly promotes pcNSCs proliferation although the underlying mechanisms and its proliferative effect in the brain remain unclear. The purpose of the present study was to clarify the possible mechanism and the effect on neurogenesis in vivo. Proteome analysis of the lysates of pcNSCs after treatment with CCC was first performed, showing that CCC up- and down-regulated 81 and 248 proteins, respectively. According to further KEGG pathway enrichment analysis, PI3K-Akt signaling was significantly enriched. Then, we checked expression of proteins related to Akt signaling including phosphorylated Akt (p-Akt) in pcNSCs by western blot analysis. Expression of p-Akt in pcNSCs treated with CCC was higher than that in vehicle-treated control. We also performed intrahippocampal injection of CCC in mice to examine whether or not CCC affects neurogenesis in hippocampal dentate gyrus (DG), which highly contains NSCs. Area of newborn neuron marker doublecortin-positive cells in the hippocampal DG injected with CCC was significantly higher than that in control. These results suggest that CCC has a potential to promote proliferation of NSCs in the brain, and Akt signaling is one of the possible signaling pathways involved.