The elasticity of the cell and that of the supporting extracellular matrices (ECMs) in tissue are correlated. In some cases, the modulus of the ECM varies with a high spatial gradient. To study the effect of such a modulus gradient on the cell culture behavior, we proposed a novel yet straightforward method to fabricate elastomeric micropillar substrates with different height gradients, which could provide a large range of elasticity gradient from 2.4 kPa to 60 kPa. The micropillars were integrated into a microfluidic chip to demonstrate the elasticity variation, with the theoretical results proving that the elasticity of the two micropillar substrates was in the same range but with distinguished gradient strengths. Fibroblast seeded on the micropillar substrates showed migration toward the stiffer area but their elongation highly depended on the strength of the elasticity gradient. In the case of high gradient strength, cells could easily migrate to the stiffer area and then elongated perpendicularly to their migration direction. Otherwise, cells were mostly elongated in the direction of the gradient. Our results also showed that when the cell density was sufficiently high, cells tended to be oriented in the same direction locally, which was affected by both underneath pillars and cell-cell contact. The elasticity gradients could also be generated in a ripple shape, and the cell behavior showed the feasibility of using the micropillars for cell patterning applications. Moreover, the gradient pillar substrates were further used for the aggregate formation of induced pluripotent stem cells, thus providing an alternative substrate to study the effect of substrate elasticity on stem cell behavior and differentiation.