Memory consolidation is a process to stabilize short-term labile memory, generating long-term memory. A critical biochemical feature of memory consolidation is a requirement of new gene expression. Protein glycosylation is one of important post-translational modifications and plays critical roles in the protein function. The most common forms of protein glycosylation are serine- or threonine-linked (O-linked) glycosylation and asparagine-linked (N-linked) glycosylation. Our previous study has shown that the inhibition of N-linked glycosylation in the hippocampus impaired long-term contextual fear and social recognition memories. In this study, we tried to understand roles of O-linked glycosylation in learning and memory. To do this, we examined effects of a micro-infusion of PUGNAc, O-linked N-acetylglucosaminidase (O-GlcNAcase) inhibitor, into the hippocampus on contextual fear memory. Mice were micro-infused PUGNAc (3.5 µg /side) into the dorsal hippocampus immediately after the conditioning with a single footshock (0.4 mA, 2 sec), and then assessed freezing at 2 or 24 hrs after the training. Mice micro-infused PUGNAc showed significantly less freezing at the test 24 hrs, but not 2 hrs, after the conditioning compared with control group. Our observations indicated that the inhibition of O-GlcNAcase in the hippocampus impaired long-term contextual fear memory without affecting short-term memory, suggesting that hippocampal O-linked glycosylation is required for consolidation of contextual fear memory.