Abstract: 【Objective】 This study aimed to induce autotetraploid of pumpkin in China using colchicine and to perform identification and characteristics analysis， thereby providing reference for breeding new pumpkin varieties with high qua-lity， high yield， and stress resistance. 【Method】 Seedling shoot tips of the advanced inbred line DP13 of pumpkin in China were treated with 0.20% colchicine. Autotetraploids were identified through morphological observation and ploidy analysis. Comparative analysis were conducted between diploid and autotetraploid pumpkin plants， including vegetative growth traits， male flower morphology， pollen germination rate， fruit characteristics， fruit quality， antioxidant enzyme activities， and genome-wide methylation levels. 【Result】 Tetraploid pumpkin plants were successfully obtained following treatment with 0.20% colchicine solution. Compared with diploid pumpkin plants， autotetraploid pumpkin plants showed significant increase by 34.69% in stem diameter （P<0.05， the same below）， extremely significant increase by 28.57% in leaf length and 32.00% in leaf width （P<0.01， the same below）， no significant difference in internode length or leaf length/leaf width ratio（P>0.05， the same below）， and a notable increase in leaf area. Autotetraploid pumpkin plants exhibited significantly reduced male flower petal width， extremely significant increase in male flower petal length/petal width， significantly enlarged pollen grains， and significantly reduced by 61.30% in pollen germination rate. In autotetraploid pumpkin fruits，flesh thickness and fruit horizontal diameter/fruit vertical diameter were significantly increased by 16.10% and 30.87% respectively，while seed cavity diameter and fruit vertical diameter were extremely significantly increased by 24.07% and 21.19% respectively， compared with diploid pumpkin fruits. Compared with diploid pumpkin fruits， the contents of sucrose， total sugar， and soluble solids in autotetraploid fruits decreased by 14.71%， 16.60%， and 6.08% respectively， whereas water content and the contents of fructose， amylopectin， amylose， total starch， and total pectin increased by 2.85%， 3.07%， 20.50%， 9.08%， 22.03%， and 21.72% respectively， indicating an overall improvement in fruit quality. The activities of catalase （CAT） and peroxidase （POD） were simultaneously and significantly increased， reaching 43283.58 U/g and 91.12 U/g respectively. The genome-wide methylation level was significantly decreased by 16.67%. 【Conclusion】 Treatment of diploid pumpkin seedlings with 0.20% colchicine successfully generates autotetraploid pumpkin plants， which exhibit pronounced growth advantages and improved overall fruit quality， but with reduced pollen germination rate and genome-wide methylation level.