New therapy targeting the hypoxic fraction of tumors needs to be designed as this population of cells is the most resistant to radio- and chemotherapies. Hypoxia-inducible factor (HIF) mediates transcriptional responses to hypoxia by binding to hypoxia-responsive elements (HRE) in target genes. We developed a hypoxia/HIF-dependent replicative adenovirus (HYPR-Ad) to target hypoxic cells. HYPR-Ad displays hypoxia-dependent E1A expression and conditional cytolysis of hypoxic but not normoxic cells. This work provides proof-of-principle evidence that an attenuated oncolytic adenovirus that selectively lyses cells under hypoxia can be generated. This therapeutic approach can be used to treat all solid tumors that develop hypoxia, regardless of their tissue origin or genetic alterations.