Recombinant adenovirus (Ad) vectors based on Ad type 5 have been widely used for gene transfer experiments. Conventional Ad type 5 vectors have a narrow range of tropism and are limited by the size of the transgene that can be packaged. To overcome these limitations, we previously developed an Ad vector (Ad5/35 vector) containing a chimeric Ad type 5 and 35 fiber protein. In the current study, we evaluated the ability of the Ad5/35 vector to transfer genes into human trophoblast cell lines (JAR, JEG-3 and BeWo cells), which are used as in vitro models of human placenta. We compared the gene transfer efficiency of Ad5/35 to that of conventional Ad vector. We found that expression of CD46, which are receptors for Ad5/35 vector, are higher than that of coxsackievirus and adenovirus receptor in all 3 trophoblast cell lines, as determined by flow cytometry. Next, we compared the transducing activity of Ad5 vector and Ad5/35 vector that each expressed luciferase as a reporter gene. Ad5/35 vector had greater gene transfer activity than the conventional Ad vector in all 3 trophoblast cell lines (1.82-fold in JAR cells, 5.37-fold in BeWo cells, 6.11-fold in JEG-3 cells). Thus, Ad vector that contains chimeric type 5 and 35 fiber protein can be a powerful tool for gene transfer experiments in human trophoblast cell lines.