The major functional properties of lactoferrin (LF) samples before and after high‐temperature short‐time (HTST: 72°C/15 s, 85°C/15 s and 95°C/15 s) treatment were investigated by turbiscan stability index, circular dichroism spectroscopy and other methods. The lower the LF iron saturation, the higher the iron binding, antibacterial and antioxidant capacity, but more unstable after HTST treatment. 40%‐LF had the best physical and structural stability, but lower antibacterial and antioxidant capacity. Upon HTST treatment, the physical stability, antibacterial and antioxidant properties of LF samples were significantly reduced, especially for 95°C/15 s. Of note, LF with iron saturation higher than 15% was resistant to HTST treatment.