Royal jelly (RJ) produced by worker honeybees is the sole food for the queen bee throughout her life as well as the larvae of worker bees for the first 3 days after hatching. Supplementation of RJ in the diet has been shown to increase spatial memory in rodents. However, the key constituents in RJ responsible for improvement of cognitive function are unknown. Our objective was to determine if the major royal jelly proteins (MRJPs) extracted from RJ can improve the spatial memory of aged rats. The spatial memory assay using the Morris water maze test was administered once to rats after a 14-week feeding. Metabolomics analysis based on quadrupole time-of-flight mass spectrometry was conducted to examine the differences in compounds from urine. Aged male rats fed MRJPs showed improved spatial memory up to 48.5% when compared to the control male aged rats fed distilled water. The metabolite pattern of the MRJPs-fed aged rats was regressed to that of the young rats. Compounds altered by MRJPs were mapped to nicotinate and nicotinamide metabolism, cysteine taurine metabolism, and energy metabolism pathways. In summary, MRJPs may improve spatial memory and possess the potential for prevention of cognitive impairment via the cysteine and taurine metabolism and energy metabolism pathways in aged rats.