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Over one billion people globally are vitamin D (VD) deficient. Studies on the biological roles of VD are numerous but very little on the stomach. This project aims to understand how gastric homeostasis is affected by VD deficiency caused by prolonged exposure to darkness alone or combined with VD deficient diet. Three groups of C57/BL6 mice were subjected to different light exposure conditions and diets for 12 months (<i>n</i> = 8–12/group): control—12 h/12 h light/dark <b><i>SDL</i></b> (<b><i>S</i></b>tandard <b><i>D</i></b>iet/<b><i>L</i></b>ight), 24 h dark <b><i>SDD</i></b> (<b><i>S</i></b>tandard <b><i>D</i></b>iet/<b><i>D</i></b>ark), and 24 h dark <b><i>VDD</i></b> (<b><i>VD</i></b> deficient diet/<b><i>D</i></b>ark). Stomach samples were collected for different multi-label lectin-/immuno-histochemical and qRT-PCR analyses, and the serum for LC-MS-MS. We found that the membrane VD receptor is expressed widely in the stomach when compared to nuclear VD receptors. Compared to <i>SDL</i>, <i>VDD</i> mice developed mucous cell expansion with increased mucins-mRNA (3.27 ± 2.73 (<i>p</i> < 0.05)) increased apoptotic cells, 15 ± 7 (<i>p</i> ≤ 0.001)); decreased cell proliferation, 4 ± 4 (<i>p</i> < 0.05)) and decreased acid secretion 33 ± 2 μEq/kg (<i>p</i> ≤ 0.0001)). Interestingly, mice exposed to full darkness developed mild VD deficiency with higher VD epimer levels: 11.9 ± 2.08 ng/mL (<i>p</i> ≤ 0.0001)), expansion in zymogenic cell number (16 ± 3 (<i>p</i> ≤ 0.01)), and a reduction in acid secretion (18 ± 2 μEq/kg (<i>p</i> ≤ 0.0001)). In conclusion, changes in light exposure or VD levels have serious physiological effects on the gastric mucosa, which should be considered during the management of gastric disorders.