Background: Sepsis is a progressive inflammatory response with serious condition and high mortality rate. Gram-negative bacteria are the most common pathogens that cause inflammatory sepsis by promoting the release of pro-inflammatory cytokines via the activation of NF-κB-dependent pathway. Severe sepsis can cause cardiac dysfunction and increases the chance of death. Artemisia lactiflora is a Chinese-origin medicinal plant that contains phenolics and flavonoids with probable anti-inflammatory activities. Therefore, the purpose of this study was to investigate the anti-inflammatory activity of A. lactiflora leaves extract on a cardiac sepsis model. Methods: Leaves of A. lactiflora were extracted and fractioned with solvents of different polarities. The cytotoxicity of extract and fractions was evaluated and calculated by neutral red assay and curve fitting analysis. Sepsis condition of cardiomyoblasts (H9c2) was stimulated with live culture of Escherichia coli (O157:H7). Anti-inflammatory activity of A. lactiflora was observed by treating cardiomyoblasts with the extract and fractions in the presence of E. coli. Expression of pro-inflammatory genes (TNF, IL6) and secretion of pro-inflammatory cytokines (TNF-α, IL-6) were measured by RT-qPCR and ELISA. Results: Co-treatment of A. lactiflora extract and all fractions significantly downregulated expression of TNF and IL6 as well as decreased secretion of TNF-α and IL-6, p < 0.0001, compared with E. coli-stimulated cardiomyoblasts. The butanol fraction had the highest potency in reducing pro-inflammatory cytokines secretion of cardiomyoblasts under sepsis condition. As a result, A. lactiflora leaves extract demonstrated therapeutic potential in the cardiac sepsis model by alleviating inflammatory response. The plant can potentially be developed as an alternative medicine for inflammation and sepsis.