Abstract: The medium stored in the hydrogen storage cylinder is high-pressure hydrogen, which will cause serious safety accidents once a leak occurs. The valve of hydrogen storage cylinders typically employ O-rings for sealing. Due to direct exposure to high-pressure hydrogen permeation and pressure, the sealing performance may change, thereby affecting the overall sealing effect of the valve. Therefore, with the goal of improving the sealing performance and service life of the valve groove structure of Type IV hydrogen storage cylinders, this paper adopts a self-developed hydrogen test device to simulate the influence of hydrogen on the O-ring under actual working conditions and uses the test results of the compressive properties of the O-ring to fit the constitutive equation of the material. In addition, the simulation analysis of the grooves of the bottle valve is also carried out to study the influence of varying groove bottom angles on the maximum equivalent stress. It is found that the maximum equivalent stress of the O-ring before and after the hydrogen test is the smallest when the groove bottom angle is 100°, and the maximum equivalent stress of the O-ring at this angle is reduced by 21.0% compared to that prior to the hydrogen test, whereas the stress reduction is only 14.3% when adopting the conventional groove design. Meanwhile, under the influence of hydrogen, the maximum equivalent stress of the O-ring is reduced by 6.6% when adopting a ramp groove structure with a bottom angle of 100°, compared to the conventional groove design of bottle valve. And the stress concentration distribution becomes more uniform.