Abstract: In this study, 5-methyltetrahydrofolate and casein were selected as the research objects. In an in vitro digestion model, particle size, zeta potential, bioaccessibility, and microstructure were measured to explore the influence of casein under different conditions on the digestion characteristics of 5-methyltetrahydrofolate. Meanwhile, using a rat pharmacokinetic method, the impact of different doses of casein on the absorption kinetics of 5-methyltetrahydrofolate was investigated. The results showed that during in vitro digestion, when the concentration of casein was 3%, casein was homogenized, intestinal pH was 6, and digestion time was 5 hours, the most significant effects of casein on the particle size, zeta potential, and bioaccessibility of 5-methyltetrahydrofolate were observed (P<0.05). Compared with the 5-methyltetrahydrofolate control group, the particle size decreased to 436.00~1465.33 nm, the absolute value of zeta potential increased to 18.58~30.64 mV, and the bioaccessibility increased to 55.96%~87.06%. The microstructure suggested that casein could bind with 5-methyltetrahydrofolate, reduce its degradation in the gastrointestinal tract, and ensure its more efficient transportation to the intestine. During in vivo absorption, the low, medium, and high dose groups of casein significantly increased the bioavailability of 5-methyltetrahydrofolate (P<0.05), with 43.18%, 50.21%, 60.24%. In conclusion, casein promotes the digestion and absorption of 5-methyltetrahydrofolate. This study provides a theoretical basis for the application of 5-methyltetrahydrofolate in dairy products.