Calcific aortic stenosis (CAS) is more prevalent, occurs earlier, progresses faster and has worse outcomes in patients with chronic kidney disease (CKD). The uremic toxin indoxyl sulfate (IS) is powerful predictor of cardiovascular mortality in these patients and a strong promoter of ectopic calcification whose role in CAS remains poorly studied. The objective of this study was to evaluate whether IS influences the mineralization of primary human valvular interstitial cells (hVICs) from the aortic valve. Primary hVICs were exposed to increasing concentrations of IS in osteogenic medium (OM). The hVICs’ osteogenic transition was monitored by qRT-PCRs for BMP2 and RUNX2 mRNA. Cell mineralization was assayed using the o-cresolphthalein complexone method. Inflammation was assessed by monitoring NF-κB activation using Western blots as well as IL-1β, IL-6 and TNF-α secretion by ELISAs. Small interfering RNA (siRNA) approaches enabled us to determine which signaling pathways were involved. Indoxyl-sulfate increased OM-induced hVICs osteogenic transition and calcification in a concentration-dependent manner. This effect was blocked by silencing the receptor for IS (the aryl hydrocarbon receptor, AhR). Exposure to IS promoted p65 phosphorylation, the blockade of which inhibited IS-induced mineralization. Exposure to IS promoted IL-6 secretion by hVICs, a phenomenon blocked by silencing AhR or p65. Incubation with an anti-IL-6 antibody neutralized IS’s pro-calcific effects. IS promotes hVIC mineralization through AhR-dependent activation of the NF-κB pathway and the subsequent release of IL-6. Further research should seek to determine whether targeting inflammatory pathways can reduce the onset and progression of CKD-related CAS. Display omitted •Indoxyl-sulfate (IS) promotes hVIC osteogenic transition and mineralization.•HVICs express the aryl hydrocarbon receptor (AhR).•IS activation of AhR promotes the NF-κB pathway and subsequent release of IL-6.•Targeting NF-κB activation or IL-6 secretion reduces IS-induced hVIC mineralization.•IS-induced hVIC inflammation might be a key driver of CKD-related aortic stenosis.