Tamoxifen (TMX) is a selective estrogen receptor modulator that is used as an estrogen receptor antagonist for the treatment and prevention of breast cancer. Whether TMX has antagonist activities in the human brain is less clear and its effects on cognitive function have not been experimentally explored. This study examined how TMX affected cognitive performance in older women using a model of anticholinergic drug-induced cognitive dysfunction. Twenty-one postmenopausal women were administered 20 mg of oral TMX or placebo for 3 months. Participants then took part in five drug challenges using the anticholinergic antinicotinic agent mecamylamine (MECA) and antimuscarinic agent scopolamine (SCOP) and were tested on a comprehensive battery including tasks of attention and psychomotor function, verbal episodic memory, and spatial navigation. After a 3-month placebo washout, participants were then crossed over to the alternate treatment and repeated the drug challenges after 3 months. Compared with placebo treatment, TMX significantly attenuated the impairment from cholinergic blockade on tasks of verbal episodic memory and spatial navigation, but effects on attentional/psychomotor tasks were more variable. Analysis by APOE genotype showed that APO varepsilon4+ women showed a greater beneficial effect of TMX on reversing the cholinergic impairment than APO varepsilon4- women on most tasks. This study provides evidence that TMX may act as an estrogen-like agonist to enhance cholinergic system activity and hippocampally mediated learning.