The economic implications of high-speed rail (HSR) cannot be overlooked in China. This paper studies the impact of HSR on the advancement and rationalization of industrial structure and the tertiary industry aggregation through theoretical derivation and multi-period difference-in-differences (DID) by improving the theoretical framework and empirical methods according to the characteristics of China's HSR and economic development. From analyses on urban heterogeneity and inter-industry spillover effects, the transmission mechanism and expressions of the industrial structure are also discussed. The findings show that HSR promotes tertiary industry aggregation and contributes to the transformation of the industrial structure from the primary to secondary and tertiary industry sectors, as well as realizing industrial structure advancement but irrationalization. Furthermore, HSR has a more significant influence on tertiary industry aggregation in large cities and high-density cities. Additionally, the aggregation of the transportation, warehousing, and postal sectors has been reduced, with a significant spillover effect on neighboring cities, proving the siphon effect and conduction mechanism, resulting in a structural shift in the tertiary industry, from basic to advanced sectors. The movement of human resources is a key mediator in the economic impact of HSR.