Objective: To investigate the implementation of sex education and its influencing factors among preschool teachers in Luzhou City, Sichuan Province, so as to provide insights into promoting the development of sex education, physical and mental health for preschool children. Methods: In January 2022, preschool teachers were sampled in 24 kindergartens in Longmatan District, Naxi District and Hejiang County of Luzhou City using the stratified random cluster sampling method. Demographic information, knowledge, attitudes, implementation of sex education, as well as difficulties, support, and needs in implementing sex education among preschool teachers were collected through questionnaire surveys. Factors affecting practice of sex education by preschool teachers were analyzed using a multivariable logistic regression model. Results: A total of 461 preschool teachers were included, including 456 females (98.92%), and had a median age of 28.00 (interquartile range, 9.50) years. The proportions of preschool teachers with good and pass of knowledge about sex education for preschool children were 10.20% and 57.48%, respectively. The proportions of preschool teachers with positive and general attitudes towards sex education were 28.20% and 36.44%, respectively. A total of 343 preschool teachers had implemented sex education (74.40%). A lack of a relaxed social environment was the main difficulty in carrying out sex education for preschool children (76.14%), while support from parents of preschool children was the primary need for conducting sex education activities (75.27%). Multivariable logistic regression analysis showed that age (>30 years, OR=2.218, 95%CI: 1.256-3.917), kindergarten type (public, OR=2.101, 95%CI: 1.253-3.524), attitude towards sex education (positive, OR=2.083, 95%CI: 1.036-4.187), participation in activities related to sex education (OR=6.355, 95%CI: 3.362-12.015), and communication with parents about sex education (OR=5.399, 95%CI: 3.152-9.248) were influencing factors for practice of sex education by preschool teachers. Conclusions The main difficulty in the practice of sex education for preschool children by preschool teachers in Luzhou City is a lack of a relaxed social environment, and there is a high demand for support from parents. Age, kindergarten type, attitude towards sex education, participation in activities related to sex education, and communication with parents about sex education are associated factors.