Abstract Background Epidemiological studies have shown associations between vitamin D, mental health and glucose homeostasis in the elderly. Causal evidence, however, is still lacking. Objective The objective of this study was to investigate the importance of vitamin D in the prevention of emotional disturbances and cognitive decline in aging C57BL/6 mice, with pre-diabetes type II as potential effect modifier. Methods Mice were exposed to one of four diets from 10 months till 24 months of age: low fat vitamin D adequate (LFD), LF vitamin D deficient (LF), moderate fat vitamin D adequate (MFD), and MF vitamin D deficient (MF). The MFD/MF diet was applied to induce a condition resembling pre-diabetes type II. Behavior was assessed twice in the same group of mice at 6-8 and at 22-23 months of age using the Open Field Test (OFT), Elevated Plus Maze (EPM), Object Recognition Test (ORT) and the Morris Water Maze (MWM). Results We successfully induced vitamin D deficiency in the LF/MF mice. Moreover, fasting glucose and fasting insulin levels were significantly higher in MFD/MF mice than in LFD/LF mice. A significant aging effect was observed for most behavioral parameters. A MF(D) diet was shown to delay or prevent the age-related increase in emotional reactivity in the EPM. No effect of vitamin D or vitamin D*fat on behavioral outcomes was measured. Conclusion Aging significantly affected emotional reactivity and cognitive performance. Although other studies have shown effects of vitamin D on emotional reactivity and cognitive performance in mice, these findings could not be confirmed in aged C57BL/6 mice in this study.