After systemic administration, adenoviral vectors (AdVs) are sequestered in the liver and adrenal glands. Adenoviral vector transduction has been shown to cause cytopathic effects on human hepatocytes and to induce an inflammatory response, whereas the effect of AdVs on human adrenocortical cells has never been investigated. In this study, human adrenocortical carcinoma cell lines and primary cell cultures were used to assess the effects of wild-type adenovirus (Ad5WT) and E1/E3-deleted AdVs on cell proliferation and steroidogenesis. Ad5WT could efficiently replicate in adrenocortical cells, leading to S phase induction, followed by cell death, whereas high titer AdVs transduction had only mild effects on adrenocortical cell proliferation, with accumulation of cells in G2/M. Both AdVs and Ad5WT induced expression of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha, but, most importantly, they led to a marked and dose-dependent increase of cortisol and other steroid hormone production and consistently modulated expression of key steroidogenic enzymes and regulators of steroidogenesis. This effect, which was already apparent at 6 h post-infection, probably represented a response to adenoviral entry and/or early phases of infection. The result of this study contribute to the understanding of host response to adenoviral vector administration.