miR-214 has been recently found to be significantly downregulated in calcified human aortic valves (AVs). ER stress, especially the ATF4-mediated pathway, has also been shown to be significantly upregulated in calcific AV disease. Since elevated cyclic stretch is one of the major mechanical stimuli for AV calcification and ATF4 is a validated target of miR-214, we investigated the effect of cyclic stretch on miR-214 expression as well as those of ATF4 and two downstream genes (CHOP and BCL2L1). Porcine aortic valve (PAV) leaflets were cyclically stretched at 15% for 48 h in regular medium and for 1 week in osteogenic medium to simulate the early remodeling and late calcification stages of stretch-induced AV disease, respectively. For both stages, 10% cyclic stretch served as the physiological counterpart. RT-qPCR revealed that miR-214 expression was significantly downregulated during the late calcification stage, whereas the mRNA expression of ATF4 and BCL2L1 was upregulated and downregulated, respectively, during both early remodeling and late calcification stages. When PAV leaflets were statically transfected with miR-214 mimic in osteogenic medium for 2 weeks, calcification was significantly reduced compared to the control mimic case. This implies that miR-214 may have a protective role in stretch-induced calcific AV disease.