Chronic hypoxia (CH) produces a time-dependent increase of resting ventilation and the hypoxic ventilatory response (HVR) that is called ventilatory acclimatization to hypoxia (VAH). VAH involves plasticity in arterial chemoreceptors and the central nervous system (e.g. nucleus tractus solitarius, NTS) but signals for this plasticity are not known. We hypothesized hypoxia inducible factor 1-a (HIF-1a), an O2-sensitive transcription factor, is necessary in the NTS for normal VAH. We tested this in two mouse models using loxP-Cre gene deletion. First, HIF-1a was constitutively deleted in CNS neurons (CNS-HIF-1a-/-) by breeding HIF-1a floxed mice with mice expressing Cre-recombinase driven by CaM Kinase II promoter. Second, HIF-1a was deleted in NTS neurons in adult mice (NTS-HIF-1a-/-) by microinjecting adeno-associated virus that expressed Cre-recombinase in HIF-1a floxed mice. In normoxic control mice, HIF-1a deletion in the CNS or NTS did not affect ventilation nor the acute HVR (10–15 min hypoxic exposure). In mice acclimatized to CH for one week, ventilation in hypoxia was blunted in CNS-HIF-1a-/- and significantly decreased in NTS-HIF-1a-/- compared to control mice (p < 0.0001). These changes were not explained by differences in metabolic rate or CO2. Immunofluorescence showed that HIF-1a deletion in NTS-HIF-1a-/- was restricted to glutamatergic neurons. The results support that HIF-1a is a necessary signal for VAH and the previously described plasticity in glutamatergic neurotransmission in the NTS with CH. HIF-1a deletion had no effect on the increase in normoxic ventilation with acclimatization to CH indicating this is a distinct mechanism from the increased HVR with VAH.