Purpose: The primary objective of this study was to evaluate knowledge and behavior of medication use among guardians of left-behind children (LBC) and non-left-behind children (NLBC). Methods: A cross-sectional study was conducted in Chengdu, the major city of southwestern China from May 2020 to August 2020. A logistic regression model was conducted to assess medication-related knowledge and behavior of guardians between the LBC group and NLBC group, adjusted for confounders. Stratified analysis was further performed. Results: The overall mean scores for knowledge and for behavior were 20.22 (standard deviation = 4.472) and 15.77 (standard deviation = 3.604), respectively. No significant difference was found in medication-related knowledge and behavior scores between LBC and NLBC guardians (P > 0.05). A significant difference was only observed after adjusting for past medical history and history of present illness (HPI). Conclusion: There was no significant difference in the awareness and behavior of medication use between guardians of LBC and NLBC in this study, having more contact with the doctor was an effective method of health education that could possibly improve their health literacy.