Concerning the problems of single fare, low revenue rate of passenger transport and unbalanced passenger flow in different sections of high-speed railway, an adjustment strategy of high-speed railway fare based on passenger flow assignment was proposed. Firstly, the related factors affecting passenger travel choice behavior were analyzed, and a generalized travel cost function including four indicators of economy, rapidity, convenience and comfort was constructed. Secondly, a bilevel programming model considering the maximization of revenue of railway passenger transport management department and the minimization of passenger travel cost was established, in which the upper level programming achieved the maximum revenue of high-speed railway passenger transport by formulating fare adjustment strategy, the lower-level programming took the minimum passenger generalized travel cost as the goal, and used the competition and cooperation relationship between different trains of section to construct Stochastic User Equilibrium (SUE) model, and the model was solved by Method of Successive Averages (MSA) based on the improved Logit assignment model. Finally, the case study shows that the proposed fare adjustment strategy can effectively balance the section passenger flow, reduce passenger travel cost and improve passenger transport revenue to a certain extent. The experimental results show that the fare adjustment strategy can provide decision support and methodological guidance for railway passenger transport management departments to optimize fare system and formulate fare adjustment schemes.