Multiple myeloma (MM) is incurable and invariably becomes resistant to chemotherapy. Although the mechanisms remain unclear, hypoxic conditions in the bone marrow have been implicated in contributing to MM progression, angiogenesis, and resistance to chemotherapy. These effects occur via adaptive cellular responses mediated by hypoxia-inducible transcription factors (HIFs), and targeting HIFs can have anti-cancer effects in both solid and hematological malignancies. Here, it was found that in most myeloma cell lines tested, HIF1α, but not HIF2α expression was oxygen dependent and this could be explained by the differential expression of the regulatory prolyl-hydroxylase isoforms. The anti-MM effects of a sequence-specific DNA-binding pyrrole-imidazole polyamide (HIF-PA), that disrupts the HIF heterodimer from binding to its cognate DNA sequences, were also investigated. HIF-PA is cell permeable, localizes to the nuclei, and binds specific regions of DNA with an affinity comparable to that of HIF transcription factors. Most of the MM cells were resistant to hypoxia-mediated apoptosis, and HIF-PA treatment could overcome this resistance in vitro. Using xenograft models, it was determined that HIF-PA significantly decreased tumor volume and increased hypoxic and apoptotic regions within solid tumor nodules and the growth of myeloma cells engrafted in the bone marrow. This provides a rationale for targeting the adaptive cellular hypoxic response of the O2-dependent activation of HIFα using polyamides.