Abstract: The objective of this research was to optimize Cucumis regeneration system, so as to make preparation for genetic transformation. The cucumber cultivar (‘Changchunmici’) cotyledon-nods were used as explants to investigate the effect of seed germination mediums, types of explants, seedling stages, phytohormone combinations and their ratios on the shoot-inducing mediums and the shoot elongation mediums. Results showed that higher efficiency of seed germination was obtained easily when the seeding seeds on 1/2MS sucrose 30 g/L agar 7 g/L, pH 5.8. The results indicated that the regeneration rate from different types of explants showed as cotyledon-nod > hypocotyl > cotyledon. The explant of cotyledons completely separated from husk but not flattened had high shoots regeneration rate than other seedling stages. Regeneration rate would reduce quickly when the cotyledons were flattened. When determining the concentration of 6-BA (6-benzyl amino purine) and ABA (Abscisic acid), the optimum of AgNO₃ was 2 mg/L. When determining the concentration of 2 mg/L AgNO₃ (2 mg/L), the best concentration combination of BA and ABA was 6-BA 1.5 mg/L ABA 0.5 mg/L. The results showed that MS 6-BA 1.5 mg/L ABA 0.5 mg/L AgNO₃ 2 mg/L medium was the optimal formula. The highest shoot regeneration frequency reached 90% and 3.5 shoots per explant. 6-BA at 0.5 mg/L markedly increased shoot elongation. The regeneration system of cucumber is successfully optimized and the robust cucumbers are obtained.