The effects of acute hypoxia have been widely studied, but there are few studies of transcriptional responses to hours of hypoxia in vivo, especially in hypoxia‐tolerant tissues like skeletal muscles. We used RNA‐seq to analyse gene expression in plantaris muscles while monitoring respiration, arterial blood gases, and blood glucose in mice exposed to 8% O2 for 2 or 6 h. Rapid decreases in blood gases and a slower reduction in blood glucose suggest stress, which was accompanied by widespread changes in gene expression. Early down‐regulation of genes associated with the extracellular matrix was followed by a shift to genes associated with the nuclear lumen. Most of the early down‐regulated genes had mRNA half‐lives longer than 2 h, suggesting a role for post‐transcriptional regulation. These transcriptional changes were enriched in signalling pathways in which the PI3K/Akt signalling pathway was identified as a hub. Our analyses indicated that gene targets of PI3K/Akt but not HIF were enriched in early transcriptional responses to hypoxia. Among the PI3K/Akt targets, 75% could be explained by a deactivation of ARE‐binding protein BRF1, a target of PI3K/Akt. Consistent decreases in the phosphorylation of Akt and BRF1 were experimentally confirmed following 2 h of hypoxia. These results suggest that the PI3K/Akt signalling pathway might play a role in responses induced by acute hypoxia in skeletal muscles, partially through the de‐phosphorylation of ARE‐binding protein BRF1. This article is protected by copyright. All rights reserved