Abstract: Due to the good extracellular matrix biomimetic properties, silk fibroin (SF) hydrogels have been extensively concerned and studied in the fields of tissue engineering scaffold and 3D cell culture. The most widely used ones among them are mild chemical crosslinked SF hydrogels. However, as the inevitable movement and self-assembly of hydrophobic segments of silk fibroin in such hydrogels, the conformation of silk fibroin transforms from random coil to the β-sheet conformation with lower energy level, which will dynamically change the basic characteristics (such as the stiffness and pore size) of the hydrogels. Consequently, unknown and non-negligible impacts on the biomedical application of the hydrogel were induced. Based on this perspective, the SF hydrogel, which was crosslinked by horseradish peroxidase in a mild manner, was chosen as model materials. The strategy of changing related crosslinking density was used to successfully regulate the rate of protein conformational transition in SF hydrogels. At the same time, the effect of protein conformational transition rate in such an SF hydrogel on the proliferation of cells adhering to its surface was investigated. Results showed that the rate of protein conformational transition in corresponding hydrogels was inversely related to the crosslinking density. The lower the crosslinking density was, the faster the conformational transition was. Meanwhile, the proliferation of mouse embryonic fibroblasts（NIH3T3） cells on the hydrogel surface was generally positively correlated with the rate of protein conformational transition in corresponding hydrogels. However, as it also existed the effects of the accompanied initial crosslinking densities, the final trend was that the total number of cells on hydrogels with medium conformational transition rate was relatively low, and that on hydrogels with the fastest conformational transition rate was relatively high.