Hyperhomocysteinemia (HHcy)—abnormally elevated plasma levels of homocysteine (Hcy)—has been associated with the development of neurodegenerative dementia, such as Alzheimer's disease (AD). This association suggests that HHcy might facilitate memory loss in the elderly. In our lab, we had revealed that HHcy is harmful to the spatial memory and induces Alzheimer-like pathological changes in rats by vena caudalis injection of Hcy for 2 weeks. Learning and memory require orchestrated regulation of both structural and functional synaptic plasticity in the hippocampus. However, the effects of HHcy on synaptic plasticity remains unclear. Using Morris water-maze, we explored spatial memory in hyperhomocysteinemic rats after a 2-week induction by vena caudalis injection of homocysteine as previous reported in our lab. For measurement of synaptic plasticity, Golgi staining was used to analyse dendritic morphology and spine density in hippocampal dentate gyrus (DG) and CA1 neurons, and perforant path to DG (PP-DG) long-term potentiation (LTP) was recorded. Immunohistochemistry and western blot were used to detect explore the memory-related proteins. We found that hyperhomocysteinemia impaired spatial memory by Morris water-maze. HHcy impaired PP-DG LTP with reduction of CREB protein level. Calcium/calmodulin-dependent protein kinase II (CaMKII), highly expressed in brain, plays a pivotal role in synaptic plasticity. We found that the ratio of pT286CaMKII/tCaMKII (activation) was significantly increased in HHcy group, suggesting Hcy overactive CaMKII might involved memory deficits. Notably, HHcy decreased dendritic branches crossings and density of the dendritic spines, with down-regulation of synapsin I, synaptophysin, GluR1, GluR2, NR2B, and NR2A protein levels. These changes still existed in 1 week and then disappeared in 4 weeks after the end of Hcy injection. We conclude that hyperhomocysteinemia impairs the synaptic plasticity by modulation synapse-associated proteins, which may underlie hyperhomocysteinemia involved memory deficits.