Polycomb ring finger oncogene BMI1 (B cell-specific Moloney murine leukemia virus integration site 1) plays a critical role in development of several types of cancers. Here, we report an inverse relationship between levels of BMI1 expression and adenovirus (Ad) progeny production. Enforced BMI1 expression in A549 cells impaired Ad progeny production. In contrast, knocking-down of endogenous BMI1 expression enhanced progeny production of a conditionally replicating Ad and wild-type Ad5 and Ad11p. Ad vectors overexpressing BMI1 were not impaired in the replication of progeny genomes and in the expression of E1A and Ad structural proteins. However, 293 cells infected by Ad vector overexpressing BMI1 contained a large proportion of morphologically irregular Ad particles. This effect was reversed in 293 cells pre-treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in parallel with the production of infectious Ad particles. Our findings suggest an inhibitory role of BMI1 in Ad morphogenesis that can be implied in Ad tropism and Ad-mediated cancer therapy.