Abstract: The Medicago sativa L. cv. Huaiyin variety of alfalfa was used to determine the physical changes and related differences in protein expressions of alfalfa in response to heat stress. Electrical conductivity, malonaldehyde content and cell activity were measured in alfalfa seedlings at 25 ℃, 30 ℃, 35 ℃ and 40 ℃ in combination with different durations of heat stress (0 h, 0.5 h, 1 h, 4 h, 12 h and 24 h). In addition, alfalfa seedlings treated at 35 ℃ and 40 ℃ for 24 h were used to analyze differentially expressed protein. The results showed that electrical conductivity of the test samples increased sharply after treatment at 35 ℃ and 40 ℃ for 0.5 h, but declined for treatment durations exceeding 1 h. However, malonaldehyde content of the samples increased for treatment durations exceeding 1 h but gradually declined after 4 h. Cell activity of test samples declined with increasing treatment duration. Screening analysis of the differences in proteins showed 27 significantly different spots after treatment at 35 ℃ for 24 h, 19 of which spots were raised and 5 were new. After treatment at 40 ℃ for 24 h, 51 significantly different spots were observed - 40 out of the 51 spots were raised and 11 were new. Secondary mass spectrum analyses indicated that 4 proteins - 20 kD chaperonin, Hsp70, Hsp23 and Hsp17, were only observed in samples at 40 ℃ for 24 hours. This suggested that the 4 proteins correlated with heat stress defense mechanisms, the samples produced more defense proteins at 40 ℃ to repair damage to alfalfa seedling membranes.