Conjunctival fiber generation is implicated in a wide spectrum of ocular diseases. Conjunctival wound healing is characterized by inflammation followed by re‑epithelialization, synthesis of new extracellular matrix (ECM), wound contraction and subconjunctival scar formation. The primary cause for the failure of glaucoma filtration surgery results from the excessive scarring of the filtering bleb. All‑trans‑retinoic acid (ATRA), a derivative of vitamin A, is a potent regulator of ECM synthesis, growth and differentiation. Following a previous study, which revealed that ATRA could inhibit transforming growth factor‑β‑induced human conjunctival fibroblast (HConF)‑mediated collagen gel contraction, the present study aimed to investigate the effects of ATRA on HConF migration, apoptosis, proliferation and ECM synthesis. To achieve this, the present study used Transwell migration, wound healing and Cell Counting Kit‑8 assays, flow cytometry and western blot analysis. In addition, the present study aimed to elucidate the mechanism of ATRA in mediating resistance to conjunctival scar formation. ATRA treatment resulted in an increased level of HConF apoptosis, reduced proliferation and migration, decreased collagen I and fibronectin expression, and decreased phosphorylation of PI3K and AKT. Thus, the present study showed a role for ATRA in inhibiting HConF migration, proliferation and ECM synthesis, and in promoting HConF apoptosis through the inhibition of the PI3K/AKT signaling pathway.