Iron and oxygen are essential substance for cellular activity in body tissues. Hypoxia-inducible factors (HIFs) can respond to available oxygen changes in the cellular environment and regulate the transcription of a series of target genes. The study was conducted to investigate the effects of iron supplementation on the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and antioxidant status in rats exposed to high-altitude hypoxia environment. Forty rats were divided into control (CON), hypobaric hypoxia (HH), and hypobaric hypoxia plus ferrous sulfate (FeSO4) (9.93 mg/kg body weight (BW)/day) (HFS) and hypobaric hypoxia plus iron glycinate chelate (Fe-Gly) (11.76 mg/kg BW/day) (HFG) groups. Results showed that Fe-Gly effectively alleviated weight loss and intestinal mucosa damage induced by hypobaric hypoxia, whereas FeSO4 aggravated hypobaric hypoxia-induced weight loss, liver enlargement, spleen atrophy, and intestinal damage. Iron supplementation decreased liver superoxide dismutase (T-SOD) and catalase (CAT) activity (P < 0.01) and increased iron concentration in the liver compared to HH group (P < 0.001). Moreover, Fe-Gly upregulated liver transferrin expression in messenger RNA (mRNA) level (P < 0.05) and downregulated serum erythropoietin (EPO) concentration (P < 0.01) and liver HIF-1α expression level (P < 0.05 in mRNA level; P < 0.001 in protein level) compared to HH group. The study indicated that FeSO4 supplementation at high altitudes aggravated the oxidative damage of tissues and organs that could be mediated through production of malondialdehyde (MDA) and inhibition antioxidant enzyme activities. Fe-Gly can protect hypobaric hypoxia-induced tissues injury. Moreover, iron supplementation at high altitudes affected HIF-1α-mediated regulating expression of targeting genes such as EPO and transferrin. The study highlights that iron supplementation under hypobaric hypoxia environment has possible limitation, and efficient supplementation form and dosage need careful consideration.