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In order to improve the light stability of vitamin D3 (VD3), the soy protein isolatevitamin (SPI-VD3) nanoparticles were prepared by high pressure homogenization. The influences of homogenization times on the SPI structure and VD3 light stability in SPI-VD3 nanoparticles were investigated. The results showed that when the times of high pressure homogenization was 2, compared with the sample without high pressure homogenization, the loading efficiency of SPI-VD3 nanoparticles was increased to 27.7%; the average particle size was reduced from 145.20nm to 82.00nm, the turbidity was gradually decreased and the particle size distribution was more uniform. Two times highpressure homogenization can increase the hydrophobicity of the surface of the SPI-VD3 nanoparticles and enhance the fluorescence intensity of the endogenous fluorescence spectrum. The results of Fourier transform infrared spectroscopy showed that the secondary structure of SPI-VD3 nanoparticles was changed after high pressure homogenization. When the number of homogenization times did not exceed 2, αhelix and βfold were gradually changed into βturn. When the number of times were 3 or 4, the sample may undergo insoluble aggregation. After twice highpressure homogenization, the photostability of VD3 in the sample was also improved. Compared with the VD3 control alone, the remaining amount of VD3 was increased to 166.6% after four hours of UV irradiation. Proper highpressure homogenization was a useful method for making SPI-VD3 nanoparticles with improved VD3 photostability. 