Ascorbic acid (AsA) was impregnated in strawberries under vacuum (0.09 MPa) and atmospheric pressure to compare the impregnation rate and stability of folate and freshness of strawberries after impregnation. The texture and folate content in strawberries impregnated with AsA during a 8-day cold storage were also investigated. The results indicated that vacuum impregnation resulted in a faster mass transfer rate (k2 = 0.01) with a higher AsA concentration (1.24 mg/g). The folate content in strawberries was 100–128 μg/100 g; the main types of folate were 5-methyltetrahydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, and folate degradation products or synthetic substrates para-aminobenzoic acid. After 8 days of refrigeration, the tetrahydrofolate content decreased by 7.70% and 42.51% in the vacuum impregnation and control groups, respectively. The para-aminobenzoic acid content in the vacuum impregnation and control groups increased by 57.13% and 77.89%, respectively. No significant change was observed in the 5-10 methylenetetrahydrofolate content (p>0.05). The 5-methyltetrahydrofolate content in the vacuum impregnation and control groups increased by 24.47% and 12.67% on day 4, respectively. Microstructure analysis indicated that after 8 days of refrigeration, hardness of strawberries in the vacuum impregnation group (92.60 N) was significantly higher than that in the control group (57.20 N). Therefore, vacuum AsA impregnation inhibited the degradation of tetrahydrofolate (p<0.05), thereby enhancing the stability of folate in cold storage and protecting the texture of strawberries. This study provides a theoretical basis and reference for improving the stability of folate in strawberries.