The aim of this study was to evaluate the effects of microneedling (MN) combined with an injectable hyaluronic acid (HA) gel on soft tissue regeneration in a rat gingival recession model. In this split-mouth study, seven Sprague-Dawley rats with artificially created gingival recession areas were randomized for treatment with MN+HA or HA. HA was injected in the gingival recession area of the right maxillary molar and MN+HA was performed in the gingival recession area of the left maxillary molar of rats, both 3 times, 7 days apart. At 2 weeks after the last treatment, each rat was euthanized and the surgical areas, including the maxillary first molar and periodontal tissue, were excised and evaluated by stereomicroscopy, histochemistry and enzyme-linked immunosorbent assay. Physical parameters representing periodontal soft tissue regeneration, including the vertical distance from the lowest point of the gingival margin to the crown margin along the long axis of the tooth, the gingival height and the gingival thickness, were significantly improved in the MN+HA group compared with the HA group. The MN+HA group showed a significant increase in the expression of Type 1 collagen compared with the HA group. Thus, this study demonstrates for the first time that the application of MN combined with a HA gel, a nonsurgical therapy, in a rat animal model of gingival recession improves the degree of gingival recession, increases gingival thickness and promotes periodontal soft tissue regeneration. Secondly, the effect of MN combined with the HA gel injection to promote periodontal soft tissue regeneration was significantly better than the HA gel injection alone, which suggests that the participation of MN effectively promotes periodontal soft tissue regeneration.