Exercise is known to increase the content and function of mitochondria, which are critical in cellular metabolism. Both a single session of high-intensity interval exercise (HIIE) and transient hypoxia alone can induce mitochondrial biogenesis. However, their combined effect on mitochondrial biogenesis has not been fully explored. In the current study, we combined HIIE with simulated hypoxia (3200m, oxygen fraction of 0.144) to investigate their influences on the genes and proteins related to mitochondrial biogenesis. Ten healthy males (age, 28 ± 5; BMI, 26.0 ± 3.4) completed three HIIE sessions in random orders: HIIE in hypoxia (HY, 88.4% of peak oxygen uptake (VO2peak)), HIIE in normoxia matched for relative intensity to hypoxia (NR, 88.7% of VO2peak), HIIE in normoxia matched for the absolute intensity to hypoxia (NA, 74.1% of VO2peak). Skeletal muscle samples were collected before, immediately, 3 hours and 24 hours post-exercise. HY and NR similarly increased the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) (by 606% and 480%, respectively), Hypoxia-inducible factor 1-alpha (HIF1α) (by 67% and 45%, respectively), heat shock protein 70 (HSP70) (by 237% and 314%, respectively) and vascular endothelial growth factor (VEGF) ) (by 214% and 268%, respectively), 3 hours after exercise (p<0.05). There was no significant change observed in the measured genes following NA at the measured time points (p>0.05). Total and phosphorylated level of p38 mitogen-activated protein kinase and AMP-activated protein kinase, as well as the total PGC1α protein content, did not change after the 3 exercise interventions. In conclusion, a single session of HIIE in hypoxia upregulated the expression of mitochondrial biogenesis genes, which was comparable to HIIE in normoxia matched for relative intensity to hypoxia. However, HIIE in normoxia matched for the absolute intensity to hypoxia does not provide enough stimulus for the genes associated with mitochondrial biogenesis.