Abstract: In order to develop a cheap and efficient culture system of Lactobacillus rhamnosus, walnut pulp was used to replace the nitrogen source in MRS medium. Taking cell growth as the indicator, the cultivation system was optimized in aspects of walnut pulp particle size, solid-liquid ratio, dosage, lipid removing treatment, and the shaking speed during the cultivation of Lactobacillus rhamnosus. The antibacterial activity and antioxidant activity of cell-free culture supernatant were also tested in all conditions. The obtained cells were also subjected to selenium enrichment treatment and then fed to mice for 3 d before they were exposed to simulated low-pressure hypoxia condition (altitude 5000 m) for 6 h to evaluate their anti-hypoxia potential. As results, complete replacement of the nitrogen source (beef extract and peptone) of MRS medium with the lipid-free walnut pulp filtered by 120 mesh screen at the solid-liquid ratio of 1:10 (g/mL) could reduce the cost of MRS medium by 70% and increase the cell yield by 2.5 times (from 2.6×10⁹ to 7.6×10⁹ CFU/mL), but did not affect the antibacterial activity and antioxidant activity of the fermentation broth (P>0.05). Continuous oral administration of selenium enriched bacteria for 3 days could significantly increase the hemoglobin content of mice when they were exposed to low-pressure hypoxia for 6 h (P<0.05), showing better effects than that cultivated in MRS medium. In conclusion, walnut pulp could be used to replace the nitrogen source in MRS medium for the culture of Lactobacillus rhamnosus, and effectively improve the cell growth and activity.