1,25-Dihydroxyvitamin D-3(1,25(OH)(2)D-3) is a secosteroid with antiproliferative property. It also plays a pivotal renoprotective role in diabetic nephropathy. We investigated whether 1,25(OH)(2)D-3 could inhibit the proliferation of rat mesangial cells exposed to high glucose via the DNA-damage-inducible transcript 4/mammalian target of rapamycin(DDIT4/mTOR) pathway. The cell proliferation rate and cell cycle duration were measured using cell counting kit-8 assay and flow cytometry. Protein expression was assayed by Western blot. Glucose acted as a growth factor in rat mesangial cells, promoted cell proliferation. In parallel, the protein expression of DDIT4, TSC1/TSC2, and 4E-BP1 were decreased, and Rheb, mTOR, and p70S6K were increased. Acting via the DDIT4/mTOR signaling, 1,25(OH)(2)D-3 treatment reversed these pathological changes, upregulated DDIT4, TSC1/TSC2, and 4E-BP1, downregulated Rheb, mTOR, and p70S6K. The short-term overexpression of DDIT4 inhibited the proliferation of rat mesangial cells, similar to 1,25(OH)(2)D-3 treatment. siRNA knockdown of DDIT4 suppressed antiproliferative responses to 1,25(OH)(2)D-3. These results suggest that 1,25(OH)(2)D-3 inhibits the proliferation of rat mesangial cells induced by high glucose via the DDIT4/mTOR signaling pathway.