Background: Severe esophageal stricture decreases patient's quality of life after circumferential endoscopic submucosal dissection (ESD). We aimed to evaluate the efficacy of autologous esophageal epithelial cell suspensions in preventing esophageal stricture after circumferential ESD. Methods: Twelve male mini-pigs underwent circumferential ESD and were randomized into four groups: G1 (control), G2 (esophageal stent), G3 (autologous esophageal epithelial cell suspension), and G4 (autologous esophageal epithelial cell suspension combined with esophageal stent). Post-ESD status was observed in each group, and endoscopy was performed weekly. Esophageal stents were removed 3 weeks after ESD. The esophageal stricture rates and histologic characteristics were assessed 4 weeks after ESD. Results: G1 showed the greatest weight loss (p < 0.05). Dysphagia scores were not significantly different among the groups. The esophageal mucosal stricture rates were 77.7 ± 2.9%, 74.2 ± 1.9%, 69.2 ± 3.8% and 65.9 ± 1.9% in G1-4, respectively; with the highest in G1 (G1 vs. G3, p = 0.005; G1 vs. G4, p = 0.001). The regenerated epithelium lengths were 4.408 ± 1.980 mm, 8.319 ± 0.857 mm, 11.801 ± 2.455 mm and 12.353 ± 1.111 mm in G1-4, respectively. The lowest degree of re-epithelialization was observed in G1, followed by G2, with the highest degrees in G3 and G4 (G1 vs. G3, p = 0.001; G1 vs. G4, p = 0.000). The maximum wound fibrosis thicknesses were 2.546 ± 0.389 mm, 2.136 ± 0.231 mm, 1.126 ± 0.211 mm and 1.131 ± 0.438 mm in G1-4, respectively, with higher degrees in G1 and G2 than in G3 and G4 (G1 vs. G3, p = 0.001; G1 vs. G4, p = 0.001). Conclusions: Autologous esophageal epithelial cell suspensions can promote re-epithelialization and reduce fibrosis, thus decreasing esophageal stricture severity after ESD.