Dedifferentiation has been identified as one of the causes of β-cell failure resulting in type 2 diabetes (T2D). This study tested whether increasing vitamin D receptor (VDR) expression prevents dedifferentiation of β cells in a high-glucose state in vitro. Culturing a mouse insulinoma cell line (MIN6) in a high-glucose environment decreased VDR expression. However, increased VDR following vitamin D3 (VD3) treatment improved insulin release of early-passage MIN6 and insulin index of db/- (heterozygous) islets to levels seen in normal functional islets. Treatment with VD3, its analogues and derivatives also increased the expression of essential transcription factors, such as Pdx1, MafA and VDR itself, ultimately increasing expression of Ins1 and Ins2, which might protect β cells against dedifferentiation. VD3 agonist lithocholic acid (LCA) propionate was the most potent candidate molecule for protecting against dedifferentiation, and an e-pharmacophore mapping model confirmed that LCA propionate exhibits a stabilizing conformation within the VDR binding site. This study concluded that treating db/+ islets with a VD3 analogue and/or derivatives can increase VDR activity, preventing the pathological dedifferentiation of β cells and the onset of T2D.