Previous studies have shown that testosterone attenuates stress-induced mood dysfunction and memory deterioration. However, the exact mechanism is still unknown. This study was conducted to investigate the role of long-term testosterone undecanoate on the behavioral responses in AD induced by AlCl3 + D-galactose administration and the possible alteration of the gene expression level of the Na/K ATPase pump. Adult male mice received AlCl3 in drinking water (10 mg/kg/day) and (D-gal 200 mg/kg/day), subcutaneously for 90 consecutive days, then received a single intramuscular (I.M) injection of castor oil (vehicle) on day 91, while treated groups received a single I.M injection of either low (100 mg/kg/45 days) or high dose (500 mg/kg/45 days) respectively of long-acting testosterone undecanoate on day 91. The time spent in the interaction zone during the open field test, preference index to novel objects in the novel object recognition test, spontaneous alternation percentage (SAP) in Y-maze test, and escape latency time in the Morris water maze test were used to measure the locomotor activity, long-term memory, and spatial memory in mice, respectively. The results showed that testosterone undecanoate treatment improved locomotor activity, improved preference to novel objects, improved spatial memory, and reversed anxiety and depression induced by AlCl3 + D-galactose administration in male mice, suggesting the enhancement of behavioral and memory functions brought by testosterone treatment. Moreover, testosterone undecanoate treatment did alter gene expression levels of Na/K ATPase isoforms in the brain hippocampus. In most cases, altered gene expression was significant and correlated with the observed behavioral changes. Taken together, our findings provide new insight into the effects of long-acting testosterone undecanoate administration on locomotor activity, long-term memory, anxiety, and spatial memory in male mice with Alzheimer's disease.