The aim of the present study was to investigate the possible effects of a diet containing transgenic corn (Zea mays), cultivar Bt176 expressing the insecticidal protein CryIA(b), on bacterial diversity of the cow rumen in vivo. Silage prepared with Bt176 corn and its parental non-transgenic cultivar showed no significant differences in either composition or in vivo degradability. Four cows were fed for 35 days with the different silage in a cross-over feeding experiment. The overall structure of rumen bacterial populations was analyzed using a cultivation-independent approach—ribosomal intergenic spacer analysis (RISA)—during 3 consecutive days of each feeding trial in all cows. Planktonic bacterial populations present in the rumen fluid and biofilm grown directly on the silage were analyzed separately. Statistical analysis of RISA fingerprints did not reveal significant differences in bacterial community structure between different feedings. Small differences were nevertheless observed between individuals on the structure of bacterial communities directly colonizing the silage material. A diet constituted by transgenic Bt-corn silage was demonstrated not to be substantially different from non-transgenic corn silage in terms of composition, digestibility and impact on the overall rumen microbiota.