Calcific aortic valve disease (CAVD) is caused by valve interstitial cells (VICs) initiating the thickening and calcification of valve leaflets. The present study aimed to investigate whether andrographolide (AGP) could attenuate the calcification of human valve interstitial cells (hVICs). hVICs stimulated by osteoblastic medium (OM) were treated with or without AGP. RNA sequencing was utilized to investigate changes in gene expression. Cell growth and calcification of hVICs were assessed using a CCK8 assay and Alizarin Red S staining, respectively. The expression of the two calcification-related markers, RUNX2 and ALP, were quantified by qRT-PCR, Western blotting, and immunofluorescent staining. The results indicate that hVICs treated with OM plus AGP exhibited decreased Alizarin Red S staining compared with cells treated with OM only in addition to down-regulation of ALP and RUNX2. Mappings of differentially expressed genes (DEGs) in different groups using Venn diagrams during analysis of gene expression profiles, 653 common DEGs were identified that displayed different biological functions and signaling pathways after treatment with AGP. RELA, a core factor of the NF-κB pathway was inhibited by AGP in addition to phosphorylation of AKT and ERK1/2. Thus, AGP attenuated calcification of hVICs. These results demonstrate that AGP, a promising natural product, can attenuate the process of CAVD.