Although extensive studies have evaluated the regulation effect of microenvironment on cell phenotype and cell differentiation, further investigations in the field of the cornea are needed to gain sufficient knowledge for possible clinical translation. This study aims to evaluate the regulation effects of substrate stiffness and inflammation on keratocyte phenotype of corneal fibroblasts, as well as the differentiation from stem cells towards keratocytes. Soft and stiff substrates were prepared based on polydimethylsiloxane. HTK and stem cells were cultured on these substrates to evaluate the effects of stiffness. The possible synergistic effects between substrate stiffness and inflammatory factor IL-1βwere examined by qPCR and immunofluorescence staining. In addition, macrophages were cultured on soft and stiff substrates to evaluate the effect of substrate stiffness on the synthesis of inflammatory factors. The conditioned medium of macrophages (Soft-CM and Stiff-CM) was collected to examine the effects on HTK and stem cells. It was found that inflammatory factor IL-1βpromoted keratocyte phenotype and differentiation when cells were cultured on soft substrate (∼130 kPa), which were different from cells cultured on stiff substrate (∼2 × 103kPa) and TCP (∼106kPa). Besides, macrophages cultured on stiff substrates had significantly higher expression ofIL-1βandTnf-αas compared to the cells cultured on soft substrates. And Stiff-CM decreased the expression of keratocyte phenotype markers as compared to Soft-CM. The results of our study indicate a stiffness-dependent dynamic effect of inflammation on keratocyte phenotype and differentiation, which is of significance not only in gaining a deeper knowledge of corneal pathology and repair, but also in being instructive for scaffold design in corneal tissue engineering and ultimate regeneration.