Gingival fibroblasts (GFs) play a considerable role in the maintenance of the gingival apparatus as well as in connective tissue repair. Mobility of a periodontal wound or soft tissue graft can impair connective tissue healing from the GFs. Enamel matrix derivative (EMD) is an enamel matrix protein used clinically for periodontal regeneration of intrabony defects and furcations, as well as treatment of gingival margin recessions. The goal of this project was to compare the effects of varying concentrations of EMD, with and without cyclic mechanical strain, on cellular wound fill of human GFs using an in vitro defect healing model. GFs were seeded and cultured in six-well flexible-bottomed plates. A 3-mm wound was created in the central portion of each confluent well. Three wells were treated with each EMD concentration of 0 µg/mL (control), 30 µg/mL, 60 µg/mL, or 120 µg/mL. The plates were placed in an incubator containing a strain unit to subject test plates to cyclic strain. An identical set of control plates were not flexed. Cells were examined on days 4, 8, 12, and 16. Microphotographs were taken and wound fill measurements made using image analysis software. The percent wound fill was calculated. All nonflexed plates, regardless of EMD concentration, reached > 90% defect fill at similar rates by day 16. However, in the flexed plates, EMD had a significant negative effect on defect fill. The defect fill was 55.7% for 0 µg/mL EMD, 48.2% for 30 µg/mL EMD, 36.7% for 60 µg/mL EMD, and 34.1% for 120 µg/mL EMD on day 16 for the flexed GFs. EMD, in concentrations as high as 120 µg/mL, did not significantly affect the amount of defect fill with nonflexed GFs. However, when the GFs were flexed, the addition of EMD had a significant negative effect on defect fill in a dose-dependent manner.