The widespread use of the herbicides Atrazine (ATR) has been raised attention due to its ubiquitous occurrence in the environment. As an endocrine disruptor, ATR causes reproductive, immune, nervous system toxicity in biota. In this study, we aimed to investigate metabolic profile characteristics and potential metabolic biomarker that reflects specific damage in toxic effect after ATR exposure. Hence, a metabolomics study was performed to determine the significantly affected metabolites and the reproduction and locomotion of C. elegans were investigated. Mediation analysis was used to evaluate the mediating effect of metabolites on association between ATR exposure and toxic effect. ATR (≥0.04 mg/L) caused the significant dose dependent reduction of brood size and locomotion behavior, however, the body length and width were significantly decreased only in 40 mg/L group. These results suggesting that brood size, head thrashes and body bends are more sensitive indictor to assessment ATR toxicity in C. elegans. Meanwhile, metabolomics analysis revealed that ATR exposure can induce metabolic profiles significant alterations in C. elegans. We found that 9 metabolites significantly increased and 18 metabolites significantly decreased, such as phosphatidylcholine, GMP, CDP-choline, neopterin etc. Those alteration of metabolites were mainly involved in the pathways: glycerophospholipid metabolism, glycolysis/gluconeogenesis, folate biosynthesis, glycine, serine and threoninemetabolism, pyrimidine and purine metabolism. Overall, these changes are signs of possible oxidative stress and ATP synthesis disruption modification. Mediation analysis showed a significant indirect effect of ATR exposure on brood size, via 7,8-dihydroneopterin 2',3'-cyclic-p, and phosphatidylcholine might mediate association between ATR exposure and body bends, suggesting that 7,8-dihydroneopterin 2',3'-cyclic-p and phosphatidylcholine might be potentially specificity marker for brood size and body bend respectively. This preliminary analysis investigates metabolic characteristics in C. elegans after ATR exposure, helping to understand the pathways involved in the response to ATR exposure and provide potential biomarkers for the safety evaluation of ATR.