Purpose <p>To compare the outcomes of corneal collagen cross-linking combined with lamellar keratoplasty (CXL-LK) versus LK alone in treating medically unresponsive acanthamoeba keratitis (AK).</p> Methods <p>This retrospective, non-randomized controlled clinical study included 11 eyes (CXL-LK group) and 16 eyes (LK group) at a tertiary ophthalmology center. Corneal infiltration, density of acanthamoeba cysts (AC) and pathological changes following CXL in AK patients were assessed. One-year post-operation, refractive outcomes, graft survival, corneal endothelial loss rates and AK recurrence were evaluated.</p> Results <p>The area and depth of the infiltrates were reduced following CXL in AK patients, and the density of the AC decreased. Histopathological results indicated a reduction in AC volume after CXL, with the contents appearing wrinkled and degraded. At one year postoperatively, the corneal endothelial loss rate was 10.12% in the CXL-LK group, significantly lower than the 27.43% observed in the LK group (<i>p</i> = 0.031). None of the patients in the CXL-LK group experienced AK recurrence, with an average duration of anti-amoebic treatment of 62.20 ± 50.33&#xa0;days, while a significant higher proportion of patients (31.25%) in the LK group experienced recurrence, with a significantly longer treatment duration of 182.75 ± 106.77&#xa0;days (<i>p</i> = 0.009). The graft survival rate demonstrated statistically significant differences between the two groups one year after surgery (90.9% in the CXL-LK group vs. 68.75% in the LK group, <i>P</i> &lt; 0.05).</p> Conclusion <p>CXL reduced AK infiltration, promoted AC degradation and effectively halted disease progression. CXL-LK significantly shortened the duration of anti-amoebic treatment, decreased AK recurrence and improved graft outcomes after LK.</p> Trial registration <p>ChiCTR1900026147 (2019-09-23)</p>