Objective To explore the effects of mechano-growth factor (MGF) E peptide on cell viability, migration and invasion of anterior cruciate ligament (ACL) fibroblasts. Methods ACL fibroblasts were used in this study and (1) were treated with MGF E peptide (0, 10 and 100 μg/L) for 24 h. Then, the medium was changed by 1% fetal bovine serum (FBS)-low glucose DMEM medium. Cell activity, DNA content, cell apoptosis, matrix metalloproteinases-2 (MMP-2) and MMP-9 activity, type I collagen (COL I) and type III collagen (COL III) mRNA expression were measured after continued culture for 6 and 30 h; (2) were treated with MGF E peptide (0, 5, 10, 20, 50 and 100 μg/L) for 48 h. Then, cell activity and MMP-2 activity were verified. Cell migration and invasion of ACL fibroblasts were further tested by cell scratch test and transwell assay, respectively. Results (1) At 6 h, 10 μg/L MGF E peptide significantly promoted MMP-2 and MMP-9 activities, but had no effect on cell viability, proliferation, apoptosis and mRNA expression of COL I and COL III. 100 μg/L MGF E peptide also significantly promoted MMP-2 and MMP-9 activities, as well as mRNA expression of COL I and COL III. However, it had no effect on the cell viability, proliferation and apoptosis. At 30 h, 10 μg/L MGF E peptide significantly promoted MMP-9 activity and mRNA expression of COL I and COL II, but had no effect on cell viability, proliferation, MMP-2 activity and apoptosis. 100 μg/L MGF E peptide also significantly promoted MMP-9 activity and mRNA expression of COL III, but had no effect on the cell viability, proliferation, MMP-2 activity, cell apoptosis and mRNA expression of type I collagen. (2) MGF E peptide significantly promoted migration and invasion of ACL fibroblasts with dose-dependent manner in a certain degree, which might depend on the increase of MMP-2 activity. Conclusions MGF E peptide can actively accelerate synthesis and degradation of the extracellular matrix, further promote migration and invasion of ACL fibroblasts, help ACL fibroblasts to move to the injurious site during repair process, which plays an important role in ACL tissue repair, regeneration and recovery after surgery.