Objective To investigate the role of endoplasmic reticulum stress?protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in the transdifferentiation of SD rat aortic vascular smooth muscle cells (VSMCs) to osteoblast-like cells by high glucose. Methods The primary cultured VSMCs from rat’s aortic segments were divided into 5 groups, including normal control group (5mmol/L D-glucose), mannitol group (5mmol/L D-glucose plus 25mmol/L mannitol), high glucose group (30mmol/L D-glucose), high glucose＋PERK-small interference RNA (siRNA) group (30mmol/L D-glucose plus PERK siRNA transfection), and high glucose＋scramble RNA group (30mmol/L D-glucose plus scramble RNA transfection). At 48 and 72h after the corresponding treatment, the expression of endoplasmic reticulum stress markers, including GRP78, PERK, p-PERK, eukaryotic initiation factor (eIF)2α, and p-eIF2α, osteo inductive factors such as Cbfα-1, osteocalcin, and smooth muscle cells marker α-smooth muscle antibody (α-SMA) at messenger RNA (mRNA) and protein levels were detected by RT-PCR and Western blotting, respectively. The activity of alkaline phosphatase (ALP) was measured by ALP activity testing kit. Results When compared with the normal group and the mannitol group, high glucose in presence or absence of scramble RNA transfection resulted significant increases in the mRNA levels of PERK and eIF2α (P＜0.05), while PERK siRNA transfection decreased the expression (P＜0.05). High glucose also induced high protein expression of PERK, p-PERK, eIF2α, and p-eIF2α (P＜0.05), but PERK siRNA transfection decreased the expression (P＜0.05). The activity of ALP was enhanced by high glucose (P＜0.05), but partially blocked by PERK siRNA (P＜0.05). Conclusion Endoplasmic reticulum stress PERK pathway plays a role in the transdifferentiation of VSMCs from the contractile phenotype to osteoblast phenotype induced by high glucose, and the transformation process can partially blunted by suppressing the PERK pathway.