Ophiocordyceps sinensis is a traditional Chinese herbal medicine and nutritional supplement, which is rich in functional components beneficial to health. The biosynthesis of these components is affected by environment factors, especially lipids. This study analyzed the lipid mass spectrometry characteristics and total antioxidant capacity (TAC) of O. sinensis in 5 different altitude environments and explored the important contributions of environmental factors. The pathway of glycerophospholipid metabolism and biosynthesis of secondary metabolites in O. sinensis was activated by altitude (AM) and mean annual temperature (MAT) at high altitudes. This stimulated the degradation of triglycerides (TG) and the biosynthesis of phosphatidylcholine (PC), and phosphatidylethanolamine (PE), promoted the accumulation of free radical scavenging (FRS) abilities and antioxidant components (AC), and increased its TAC. This study reflects the important role of high altitude environment on lipid metabolism and the formation of bioactive components in O. sinensis and provides a scientific basis for exploring its medicinal value and nutritional value.