Field experiment was conducted in 2004-2005 using two winter wheat cultivars differing in grain protein content, Ningmai 9′ (low protein) and Yangmai 10′ (medium protein) in order to investigate the effects of N and K rates on key enzyme activities for protein formation in flag leaves and grains, grain protein contents, and their relationships with leaf N and K nutrition status at anthesis. The results showed that free amino acid contents and GS activities in flag leaves and grains, and GPT activity in grains all increased with nitrogen and potassium supply. Compared with potassium fertilization, the effects of N on free amino acid contents and relative enzyme activities for grain protein formation were greater than those of K, with largest effects from N K treatment, and greater with Yangmai 10′ than with Ningmai 9′. The N/K ratio in flag leaves at anthesis showed quadratic curve with nitrogen supply, while decreased with potassium supply. Correlation analysis showed that the GS activity and grain protein content both had significant quadratic correlations with leaf N/K ratio at anthesis. The results suggested that the leaf N/K ratio at anthesis was the vital factor for affecting grain protein formation under different nitrogen and potassium supply.