Hypoxia is an integral characteristic of the tumor microenvironment, primarily due to the microvascular defects that accompany the accelerated neoplastic growth. The presence of tumor hypoxic areas correlates with negative outcome after radiotherapy, chemotherapy, and surgery, as hypoxia not only provides an environment directly facilitating chemo- and radio-resistance, but also encourages the evolution of phenotypic changes inducing permanent resistance to treatment and metastatic spread. Therefore, successful treatment of hypoxic cells has the potential to not only improve local control but also impact overall patient survival. Specific and selective targeting of hypoxic tumor areas can be achieved at all three steps of a gene therapy treatment: delivery of the therapeutic gene to the tumor, regulation of gene expression, and therapeutic efficacy. In this review the latest developments and innovations in hypoxia-targeted gene therapy are discussed. In particular, approaches such as hypoxia-conditionally replicating viruses, cellular vehicles, and gene therapy means to disrupt the hypoxia-inducible factor (HIF) signaling are outlined.