Currently, curative therapies for heart valve diseases do not exist, thus motivating the need for new therapeutics, regenerative and tissue-engineered valves, and further basic research into pathological mechanisms. For studying valve diseases and developing valve therapies, effective methods to manipulate gene expression in primary valvular interstitial cells (VICs), which promote calcification in disease, would be valuable. Unfortunately, there is little information reported about effective gene delivery methods for VICs. Adeno-associated virus (AAV) is a clinically proven gene delivery vector capable of transducing many cell types and tissues, but has not yet been reported to infect valvular cells. In this study, AAV serotypes 1–9 were tested for their ability to deliver a green fluorescent protein (GFP) reporter into VICs in vitro. Flow cytometry results indicate AAV2 and AAV3 are capable of transducing VICs more efficiently than other serotypes. Furthermore, transduction efficiencies can be optimized by increasing the multiplicity of infection (MOI) and using self-complementary, double-stranded genomes, yielding up to 98% successfully transduced cells. Transduction of VICs by AAV2 or AAV3 in the presence of competing soluble heparin significantly reduces delivery efficiencies, suggesting heparan sulfate proteoglycans act as the primary VIC receptors of these two serotypes. Overall, this study establishes AAV2 and AAV3 as efficient gene delivery vehicles for primary VICs. Such effective delivery vectors for valve cells may be broadly useful for numerous applications, including the study of valvular cell biology, development of valve disease therapies, and regulation of genes for tissue engineering heart valves.