Gentamicin is a widely employed antibiotic, but may reduce calcium uptake by eukaryotic cells. This study was conducted to determine whether gentamicin reduces calcification by porcine aortic valvular interstitial cells (pAVICs) grown in 2D culture, which is a common model for calcific aortic valve disease (CAVD). The presence of gentamicin (up to 0.2 mM) in the medium of pAVICs cultured for 8 days significantly lowered calcification and alkaline phosphatase content in a dose-dependent manner compared to pAVICs cultured without gentamicin. Gentamicin also significantly increased cell proliferation and apoptosis at concentrations of 0.1–0.2 mM compared to controls. Next, gentamicin was applied to previously calcified pAVIC cultures (grown for 8 days) to determine whether it could stop or reverse the calcification process. Daily application of gentamicin for 8 additional days significantly reduced calcification to below the pre-calcification levels. These results confirm that gentamicin should be used cautiously with in vitro studies of calcification, and suggest that gentamicin may have the ability to reverse calcification by pAVICs. Given the nephrotoxicity and ototoxicity of this antibiotic, its clinical potential for the treatment of calcification in heart valves is limited. However, further investigation of the pathways through which gentamicin alters calcium uptake by valvular cells may provide insight into novel therapies for CAVD.