The water soluble matrix (WSM) of pearl powder [Hyriopsis cumingii Lea (Unionidae)] was extracted, and the insoluble residue was demineralized, size-fractionated, and named as MR14 (> 14 kDa), MR3-14 (3-14 kDa), and MR3 (< 3 kDa). The effects of WSM, MR14, MR3-14, and MR3 on primary mouse oral fibroblast proliferation, collagen accumulation, matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and tissue inhibitor of metalloproteinase-1 (TIMP-1) production were tested by MTT assay, chloramine T method, gelatin zymography, and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that the WSM and MR14 could significantly (p < 0.05) promote fibroblast proliferation; all of the fractions could significantly promote collagen accumulation; MR14 significantly (p < 0.05) inhibited MMP-2 activity; and all of the fractions could significantly promote TIMP-1 production. This study has proved that the mechanism by which pearl powder promotes wound healing is partly due to its ability to stimulate fibroblast mitosis, collagen deposition, and TIMP-1 production, and the major active fraction may be MR14.