Acellular porcine corneal stroma (APCS) is a promising alternative to allografts, however its potential requires verification. To evaluate the efficacy and safety of novel cross-linked APCS transplantation for the treatment of infectious corneal disease.This prospective, multicenter, single-arm clinical trial was conducted from 2015-2019. Ninety patients with infectious corneal disease who had underwent anterior lamellar transplantation with an APCS across four centers in China were enrolled. All patients had previously received ineffective drug treatment and had a preoperative best-corrected visual acuity (BCVA) < 20/400. The primary outcome was the deblinding rate at 180 days compared to the target rate of 65%. The secondary outcomes included BCVA, corneal transparency, and epithelial healing. Safety endpoints included corneal graft rejection, blood count, liver and kidney function, immunoglobulin levels, concomitant medications, and additional treatments. Follow-up was performed for at least 6 months postoperatively.A total of 100% (90/90) and 88.9% (80/90) of the cases were included in the safety analysis and efficacy per protocol sets, respectively. Eighty percent (72/90) of patients completed 180 days of follow-up after APCS transplantation. The deblinding rate at 180 days was 90.3% (95% confidence interval 81%-96%), exceeding the target (P<0 · 001). At 180 days, 55% of the grafts were completely transparent and 82.5% of the epithelium had no defects. The log of the minimum angle of resolution vision improved from 2.19 ± 0.25 preoperatively to 1.10 ± 0.3. No serious adverse events occurred.APCS demonstrated excellent corneal transparency and epithelialization following keratoplasty, confirming its safety and efficacy. Six-month data suggest its potential as an alternative treatment for infectious corneal disease. Further long-term trials are necessary to establish its comparative effectiveness and evaluate chronic complications, offering valuable insights for future clinical practice.Copyright © 2025 The Author(s). Published by Wolters Kluwer Health, Inc.