Background <p>HIF-1α is classically activated under hypoxia, inducing metabolic adaptation, angiogenesis, extracellular matrix remodelling and immune response. However, HIF-1α activation under normoxia and the role of mechanical stimuli on hypoxic response remain incompletely characterized. This study investigates the role of mechanical stimuli in modulating HIF-1α activation under normoxia by studying the response to matrix stiffness, and the role of NF-κB pathway in this modulation.</p> Methods <p>PSCs were cultured on polyacrylamide hydrogels of defined stiffness to study HIF-1α modulation and its functional activation. The mechanosensitive regulation of NF-κB signalling, β1 integrin activation, and the RhoA-dependent MLC-2 pathway were studied. Pharmacological modulation of this mechanosensitive pathway was performed by targeting GPER as an upstream mechanoregulator.</p> Results <p>Substrate stiffness induced robust nuclear translocation and functional activation of HIF-1α under normoxia. Mechanistically, this response required the β1 integrin-induced actomyosin contractility (mediated by MLC-2), promoting the activation of NF-κB, thereby positively regulating HIF-1α mRNA levels. The intermediate NF-κB activation was required to induce functional activation of HIF-1α, coupling mechanical stimuli to inflammatory and hypoxia signalling. This signalling pathway was targeted pharmacologically by regulating actomyosin tension using a GPER agonist, which modulates both mechanosensitive NF-κB and HIF-1α activation.</p> Conclusions <p>These results reveal that substrate stiffness induces HIF-1α activation through the β1 integrin/MLC-2/NF-κB axis, implicating a mechanosensitive regulatory pathway under normoxia. These findings propose a novel feed-forward loop between matrix stiffness and HIF-1α signalling that can be pharmacologically modulated.</p>