The therapeutic utility of oncolytic adenoviruses controlled by a single, tumor-specific regulatory element may be limited by the intra- and inter-tumoral heterogeneity that characterizes many cancers. To address this issue, we constructed an oncolytic adenovirus that uses two distinct tumor-specific promoters (DF3/Muc1 and hTERT) to drive separate E1A expression cassettes, in combination with deletion of the viral E1B region, which confers additional tumor selectivity and increased oncolytic activity. The resultant virus, Adeno-DF3-E1A/hTERT-E1A, induced higher levels of E1A oncoprotein, enhanced oncolysis and an earlier and higher apoptotic index in infected tumor cells than following infection with Adeno-hTERT-E1A, which harbors a single hTERT promoter-driven E1A cassette. In isolated U251 human gliosarcoma cell holoclones (putative cancer stem cells), where DF3/Muc1 expression is substantially enriched and hTERT expression is decreased compared with the parental U251 cell population, E1A production and oncolysis were strongly decreased following infection with Adeno-hTERT-E1A, but not Adeno-DF3-E1A/hTERT-E1A. The strong oncolytic activity of Adeno-DF3-E1A/hTERT-E1A translated into superior anti-tumor activity over Adeno-hTERT-E1A in vivo in a U251 solid tumor xenograft model, where hTERT levels were >90% suppressed and the DF3/Muc1 to hTERT expression ratio was substantially increased compared with cultured U251 cells. The enhanced anti-tumor activity of the dual-targeted Adeno-DF3-E1A/hTERT-E1A was achieved despite premature viral host cell death and decreased production of functional viral progeny, which limited tumor cell spread of the viral infection. These findings highlight the therapeutic benefit of targeting oncolytic viruses to heterogeneous tumor cell populations.