PKCα has been shown to be a negative regulator of contractility and PKCα gene deletion in mice protected against heart failure. Small interfering (si)RNAs mediate gene silencing by RNA interference (RNAi) and may be used to knockdown PKCα in cardiomyocytes. However, transfection efficiencies of (si)RNAs by lipofection tend to be low in primary cells. To address this limitation, we developed an adenoviral vector (AV) driving short hairpin (sh)RNAs against PKCα (Ad-shPKCα) and evaluated its potential to silence PKCα in neonatal rat cardiac myocytes and in engineered heart tissues (EHTs), which resemble functional myocardium in vitro. A nonsense encoding AV (Ad-shNS) served as control. Quantitative PCR and Western blotting showed 90% lower PKCα-mRNA and 50% lower PKCα protein in Ad-shPKCα-infected cells. EHTs were infected with Ad-shPKCα on day 11 and subjected to isometric force measurements in organ baths 4 days later. Mean twitch tension was > 50% higher in Ad-shPKCα compared to Ad-shNS-infected EHTs, under basal and Ca²⁺- or isoprenaline-stimulated conditions. Twitch tension negatively correlated with PKCα mRNA levels. In summary, AV-delivered shRNA mediated highly efficient PKCα knockdown in cardiac myocytes and improved contractility in EHTs. The data support a role of PKCα as a negative regulator of myocardial contractility and demonstrate that EHTs in conjunction with AV-delivered shRNA are a useful model for target validation.