Recent studies demonstrate that virus-cellular receptor interactions are not the sole determinants of adenovirus (Ad) tropism. It has been shown that the fiber shaft length, which ranges from 6 to 23 β-repeats in human Ads, also influences viral tropism. However, there is no report that investigates whether artificial extension of the shaft alters the infectivity profile of Ad. Therefore, we constructed Ad serotype S (AD5) capsid-based longer-shafted Ad vectors by incorporating Ad2 shaft fragments of different lengths into the AdS shaft. We show that "longer-shafted" Ad vectors (up to 32 β-repeats) could be rescued. We also show that longer-shafted Ad vectors had no impact on knob-CAR (coxsackievirus and Ad receptor) interaction compared to wild-type Ad. Nevertheless, gene transfer efficiencies of longer-shafted Ad vectors were lower in CAR-positive cell lines compared to wild-type Ad. We suggest that artificial extension of the shaft can inhibit infectivity in the context of CAR-positive cell lines without modification of knob-CAR interaction.