Background: Gingival recession (GR) is one of the most common esthetic concerns associated with periodontal tissues. Recently, tissue engineering technology has been developed and applied in periodontology for the treatment of GR. The aim of this study is to compare the clinical efficacy of collagen membrane with or without autologous gingival fibroblasts under a coronally advanced flap for root coverage. Methods: In this split-mouth, controlled clinical study, 22 sites are selected from 11 patients with Miller Class I recessions affecting canines or premolars in the maxillary arch. One tooth in each patient was randomized to receive either a collagen membrane (CM) (control group) or a collagen membrane seeded with autologous gingival fibroblasts (CM+GF) (test group) under a coronally advanced flap. Thickness of the gingiva, GR, and percentage of root coverage (PRC) were recorded by a calibrated examiner at baseline and 3, 6, and 12 months postoperatively. Furthermore, GR and PRC were evaluated using photogrammetric analysis at baseline and 3, 6, and 12 months. Results: Both treatments resulted in a significant gain in root coverage compared with baseline. A statistically significant increase was detected in PRC in the test group compared with the control group. No significant difference was noted between the test and control sites regarding the thickness of the gingiva. Conclusions: The results indicated that CM+GF prepared by tissue engineering technology can be considered an alternative method for the treatment of Miller Class I recession defects.