Objective: To systematically evaluate the potential of Saussurea involucrata cultures (SICs) against high-altitude illness under hypobaric hypoxia and establish a progressive experimental evidence chain covering acute hypoxia tolerance enhancement and acute/chronic hypoxic injury protection. Methods: A tiered experimental strategy was employed. Key findings were derived from primary rat models of acute (5500 m, 8 h) and chronic intermittent (5500 m, 8 h/d, 4–8 weeks) hypobaric hypoxia. A mouse acute tolerance model (10,000 m lethality, closed-system endurance) provided supplementary verification. Comprehensive analyses included survival, hemorheology, multi-organ function, and core mechanistic indicators of endothelial function and oxidative stress. Diamox, Rhodiola, and Compound Danshen Dripping Pills served as positive controls. Normoxic/hypoxic blank groups served as negative controls. Results: SICs significantly enhanced acute hypoxia tolerance in mice. In the rat models, SICs demonstrated dose-dependent and selective regulation of the endothelial–oxidative stress/hemorheology axis. Specifically, it downregulated endothelin-1, upregulated nitric oxide, enhanced total antioxidant capacity, and improved chronic hypoxia-induced blood hyperviscosity. Medium doses showed consistent optimal efficacy. SICs had limited effects on macroscopic organ remodeling. Conclusions: The core protective effect of SICs lies in enhancing hypoxic tolerance and selectively modulating the interconnected pathways of endothelial function, oxidative stress, and microcirculatory health. This mechanistic profile supports its potential as a preventive or early adjuvant intervention for high-altitude illness, providing a systematic preclinical foundation for translational development.