Objective To investigate the current status, trend of changes and influencing factors of Enterobius vermicularis infection in kindergarten children in Jiangsu Province, in order to provide the scientific basis for adjustment and improvement of enterobiasis control strategy. Methods From 2019 to 2022, at least one county (city, district) was selected from the southern, central and northern parts of the Province as survey sites, from each of which two kindergartens (one rural type and one urban type) with a size over 200 children each were selected for cluster sampling the participants. In each kindergarten, no less than 200 chidren separately from senior, middle, and junior classes participated in the suvey. Anal swab method was used to detect E. vermicularis eggs by microscopy. Children’s parents underwent questionnaire survey on basic information of children, hygienic habits related to E. vermicularis infection, family background, and knowledge awareness of enterobiasis. Results A total of 15 669 children were examined in 21 counties (districts, cities), and the overall prevalence rate of pinworm infection was 0.7% in Jiangsu Province from 2019 to 2022. The annual prevalence was 1.2% (44/3 678), 0.5% (13/2 568), 0.7% (27/3 802) and 0.4% (22/5 621), with a significant decrease detected among the years (χ² = 14.46，P < 0.05). The annual prevalence of rural kindergarten was 2.1% (41/1 981), 0.2% (2/833), 1.0% (8/824) and 0.6% (19/3 271), with a significant decrease detected among the years (χ² = 17.34，P < 0.05). The annual prevalence of urban kindergarten was 0.2% (4/1 697), 0.6% (10/1 635), 0.7% (20/2 987) and 0.2% (6/2 650), with no significant difference detected among years (χ² = 0.04, P > 0.05). The overall prevalence of South Jiangsu, Central Jiangsu, and North Jiangsu from 2019 to 2022 was 1.0% (56/5 698), 0.5% (32/6 135), and 0.5% (22/4 136). The prevalence among children of the senior class was 1.8% (23/1 266), 0.8% (8/998), 1.4% (16/1 167), and 0.6% (10/1 796) from 2019 to 2022, respectively, with a significant decrease detected among years (χ² = 8.01, P < 0.05), which were higher than that among children of the middle and junior class. The prevalence among children of middle class was 1.0% (13/1 325), 0.5% (4/871), 0.5% (9/1 687), and 0.5% (10/2 063) from 2019 to 2022, respectively. The prevalence among children of junior class was 0.8% (9/1 087), 0 (0/699), 0.3% (3/948), and 0.3% (5/1 762) from 2019 to 2022, respectively. The sucking-finger habit accounted for 20.8%, 17.4%, 20.5%, and 19.4% from 2019 to 2022, respectively. The habit of sucking toys/pens accounted for 12.5%, 9.3%, 10.1%, and 12.6% from 2019 to 2022, respectively. A total of 61.9%, 76.0%, 78.5%, and 70.3% (from 2019 to 2022, respectively) parents reported that they knew enterobiasis, with a significant increase detected among the years (χ² = 202.86, P < 0.05). Being older (OR = 1.447, 95% CI = 1.186-1.764, P < 0.05), a low frequency of laundry (OR = 1.121, 95% CI = 1.002-1.225, P < 0.05), and rural precshool (OR = 0.509, 95% CI = 0.333-0.776, P < 0.05) were risk factors for pinworm infection. Conclusion The prevalence of E. vermicularis infection remained comparatively low in kindergarten children in Jiangsu Province from 2019 to 2022, with a prevalence decreasing trend. However, there exist inadequate hygienic habits in children, and parents showed low knowledge awarness rate on enterobiasis. It is suggested that the factors influencing pinworm infection in kindergarten may include children age, the frequency of washing and sunshining exposure of beddings, and the type of kindergartens.