Estrogen has a vast and complex role on higher brain abilities such as learning and memory. On the other hand, sesame oil (SO) (as a vehicle in the steroids structure) has a different effect on body function. So, in this study the effect of intrahippocampal injection of estradiol benzoate (EB) and SO on spatial learning and memory in adult NMRI male rats was investigated. The animals were anesthetized by ketamine (78 mg/kg) and xzylazine (3 mg/kg) and bilaterally microcannulated into the CA1 region of hippocampus, and after recovery they were divided into the following 4 groups (n = 7): control group (no injection) that was only trained in Y-maze. The SO test, physiological saline sham and EB test groups received 0.5 µl of SO, 0.5 µl of physiological saline and 1µg/0.5µl of EB respectively. The injections were made bilaterally into the CA1 region of hippocampus immediately before training. Then, each rat was trained in 30 trials every day for a total of 5 days with Y-maze. After one month, all of the experimental groups were tested (one session) for memory test. Statistical analysis of data using repeated measure design and least significant difference test showed a significant difference between EB and saline groups (p<0.001), but no significant difference between SO and saline groups was noted. Also, no significant difference between EB and SO groups was found out. These results indicate that EB increases and SO improves spatial learning tasks. Regarding memory test, there were no significant differences between learning results of 5th day and a month later in different groups. This may indicate that their memory has not changed. In conclusion, it seems that EB increases learning task through an interaction with cholinergic system and enhancement of synaptic plasticity in hippocampal CA1 area and SO increases learning task through its unsaturated fatty acids that may change neuronal membrane fluidity and through lecithin as a precursor of acetylcholine that may interact with cholinerigic system.