Objective To analyze the level of occupational health literacy (OHL) and mental health status in key populations in Jiangsu Province, and explore the impact of OHL on occupational stress, depressive symptoms, anxiety symptoms and insomnia symptoms. Methods A total of 11 181 workers from Jiangsu Province from April to December 2023 were selected as the research subjects using a combination of stratified cluster random sampling and probability proportional sampling method. The OHL levels of the workers, as well as their occupational stress, depression symptoms, anxiety symptoms and insomnia symptoms, were investigated using the Occupational Health Literacy Questionnaire of National Key Populations (including Occupational Health Literacy Scale, Core Occupational Stress Scale, Patient Health Questionaire-9, Generalized Anxiety Disorder 7-item Scale, and Self-Sleep Management Questionnaire). Results The OHL level of the research subjects was 41.7%, and the literacy levels of the four dimensions of occupational health legal knowledge, basic knowledge of occupational health protection, basic skills of occupational health protection, and healthy working style and behavior were 40.6%, 76.4%, 28.0% and 54.4%, respectively. The detection rates of occupational stress, depression symptoms, anxiety symptoms and insomnia symptoms were 21.8%, 17.7%, 26.4% and 29.6%, respectively. The result of multivariable logistic analysis showed that participants with adequate OHL had a lower risk of occupational stress, depressive symptoms, anxiety symptoms, and insomnia symptoms than those without adequate OHL (all P<0.01) after controlling individual and occupational confounding factors. The odds ratios and their 95% confidence intervals were 0.448(0.402-0.499), 0.664(0.593-0.742), 0.705(0.639-0.777) and 0.885(0.809-0.969), respectively. Conclusion OHL is an independent influencing factor for occupational stress, depressive symptoms, anxiety symptoms and insomnia symptoms in key populations from Jiangsu Province. Effective measures should be taken to improve the OHL level of workers, to reduce the risks of occupational stress, depressive symptoms, anxiety symptoms and insomnia symptoms.