High hydrostatic pressure (HHP) technology was used for improving the ability of β-carotene biosynthesis of Rhodotorula glutinis R68. After the treatments of five repeated cycles at 300MPa for 15 min, the barotolerant mutant PR68 was obtained. After 72 h of culture, the biomass of mutant PR68 was 21.6 g/L, decreased by 8.5% compared to the parent strain R68, but its β-carotene production reached 19.4 mg/L, increased by 52.8% compared to the parent strain R68. The result of restriction fragment length polymorphism analysis suggested that mutant strain PR68 was likely to change in nucleic acid level, and thus enhanced β-carotene production in this strain was a result of gene mutation induced by HHP treatment. HHP technology seems a promising approach for improving industrial microbes.