The mechanism of reducing dietary cation-anion difference (DCAD; Na⁺K–Cl⁻S, mmol/kg DM) to prevent hypocalcemia is not completely known. Therefore, this study was conducted to clarify the mechanism why reducing DCAD was effective for increase in plasma Ca concentration and thus for hypocalcemia prevention by detecting plasma Ca concentration and vitamin D receptor (VDR) expression level in gastrointestinal tracts (GIT) of transition mice. One hundred twenty transition mice were randomly allocated to 3 blocks with each of 40 individuals and were fed 3 diets with varying DCAD level at +300 (treatment 1, HD), +150 (control, CON), and −150 (treatment 2, LD), respectively. Ten mice for each treatment were killed to collect blood sample for plasma Ca concentration analysis and harvest GIT tissues (stomach, duodenum, jejunum, ileum, cecum, and colon) samples for VDR expression level detection at 4 time points: day of 20 (−20) and 5 (−5) before kidding, day of kidding (0), and day of 3 after kidding (+3). Data on plasma Ca was analyzed using SAS 9.3 with Proc MIXED and VDR mRNA with Proc NPar1 Way. Plasma Ca concentration (mg/dL) in LD mice (6.37) was higher than that in HD (4.43) and CON (4.98) mice for the whole and individual blood sampling time (P < 0.05). On d −20 diet LD resulted in higher VDR mRNA expression level in jejunum, ileum, and colon over diet HD (P < 0.05). On d −5 mice fed LD diet had more VDR mRNA expression level in jejunum and colon relative to HD and CON (P < 0.05). On d 0 feeding of treatment 2 had the highest VDR mRNA expression level in duodenum and colon, which was statistically higher than HD and CON (P < 0.05). On d 3 duodenum, jejunum, and colon were observed to show increased VDR mRNA expression level for LD compared with HD and CON (P < 0.05). These results indicated that reducing the DCAD upregulates VDR mRNA expression in GIT of transition mice and increases plasma Ca concentration, which explains some of the benefits of low DCAD on prevention of hypocalcemia.