Gene-modified replication-competent adenoviruses (Ads) are emerging as a promising new modality for the treatment of cancer. We have previously shown that E1B 19kDa and E1B 55kDa gene deleted Ad (Ad-ΔE1B19/55) exhibits improved tumor-specific replication and cell lysis, leading to an enhanced anti-tumor effect. In an effort to increase cancer cell-selectivity of a replicating adenovirus, we first generated eleven E1A mutant Ads (Ad-E1mt1 11) with deletion or substitution in retinoblastoma (pRb) binding sites of E1A. Of these, Ad-E1mt7 demonstrated significant improvement in cytopathic effect (CPE) and viral replication in a cancer cell-specific manner. To further enhance the cancer cell- specificity of Ad-E1mt7, Ad-ΔE1Bmt7 was generated in which both the E1B 19kDa and E1B 55kDa genes were deleted. As assessed using CPE assay, MTT assay, and immunoblot analysis for Ad fiber expression, Ad-ΔE1Bmt7 exerted marked enhancement in cancer cell-specific killing effect as well as viral replication in comparison to its comparative controls (Ad-E1mt7, Ad-ΔE1B55, Ad-ΔE1B19/55). Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-ΔE1Bmt7. In summary, we have developed an oncolytic adenovirus with significantly improved therapeutic profile for cancer treatment. Gene-modified replication-competent adenoviruses (Ads) are emerging as a promising new modality for the treatment of cancer. We have previously shown that E1B 19kDa and E1B 55kDa gene deleted Ad (Ad-ΔE1B19/55) exhibits improved tumor-specific replication and cell lysis, leading to an enhanced anti-tumor effect. In an effort to increase cancer cell-selectivity of a replicating adenovirus, we first generated eleven E1A mutant Ads (Ad-E1mt1 11) with deletion or substitution in retinoblastoma (pRb) binding sites of E1A. Of these, Ad-E1mt7 demonstrated significant improvement in cytopathic effect (CPE) and viral replication in a cancer cell-specific manner. To further enhance the cancer cell- specificity of Ad-E1mt7, Ad-ΔE1Bmt7 was generated in which both the E1B 19kDa and E1B 55kDa genes were deleted. As assessed using CPE assay, MTT assay, and immunoblot analysis for Ad fiber expression, Ad-ΔE1Bmt7 exerted marked enhancement in cancer cell-specific killing effect as well as viral replication in comparison to its comparative controls (Ad-E1mt7, Ad-ΔE1B55, Ad-ΔE1B19/55). Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-ΔE1Bmt7. In summary, we have developed an oncolytic adenovirus with significantly improved therapeutic profile for cancer treatment.