The high-speed railway expansion is a landmark and significant event in the history of China’s transportation infrastructure construction, which has profound impact on regional factor flow, economic activities and industrial spatial pattern. It is significant to probethe relationship between the opening of high-speed rail(HSR) and industrial agglomeration for the appropriateplanning of HSR infrastructureand promoting high-quality development. The article explores the spatial spillover effects and mechanisms of tertiary industry agglomeration caused by the opening of HSR, and focuses on the mechanism of the effect of knowledge spillover resulting from labor mobility on tertiary industry agglomeration based on the space-time effect of HSR. By constructing a Spatial Difference-In-Difference Model (SDID) and using the panel data of 108 prefecture-level cities in the Yangtze River Economic Belt(YREB) from 2007-2018, thepaper empirically evaluates the impact of the opening of HSR on tertiary industry agglomeration in the YREB. Further, not only the coefficient of the spatial lag term is used to judge the existence of spatial spillover effect, but also the indirect effect to measure the extent of the spatial spillover effect. Since the uneven distribution of HSR network, the disparity in economic development, and the potential differences in the effects ofthe opening of HSR in cities with different resource endowments in YREB, this paper examines the heterogeneity of the impact of HSR on the tertiary industry agglomeration by region and sub-sector as well as empirically testing the transmission mechanism of the impact of HSR on the tertiary industry from the perspective of labor transfer. The study results show that:1)HSR not only has a noticeable positive effect on tertiary industry agglomeration in cities along the YREB, but also its spatial spillover effect obviously facilitates industrial agglomeration in neighboring cities. 2)Heterogeneity test indicates that the positive effect of HSR on the level of tertiary industry agglomeration in eastern cities is higher than that of in central cities, while there is no distinct effect on tertiary industry agglomeration in western cities of YREB. In addition, the effect of HSR on different sectors varies significantly, especially on the agglomeration levels of wholesale and retail, public administration, social security and social organizations, and education.3)The test of influence mechanism suggests that the HSR expands the scope of free labor mobility resulting in knowledge spillover which brings about tertiary industry agglomeration, and that labor transfer is also an important transmission mechanism of the effect of HSR on tertiary industry agglomeration in central cities. Therefore, cities without HSR should accelerate the construction of HSR network and optimize the industrial spatial pattern; central and eastern regions should increase the undertaking of medium to high-end industries in the tertiary industry to prompt industrial upgrading; as well as western regions should give full play to the spatial reconfiguration effect of HSR on labor factors to prevent widening gap between regions. This paper introduces the spatial spillover effect into the SDID to determine whether and to what extent the spatial spillover effect of tertiary industry agglomeration caused by HSR is present. Besides, the heterogeneity under the dual influence of "siphon effect" and "radiation effect" is examined from the perspective of labor mobilityunder the "space-time compression" effect. This enriches the theoretical research on the impact of HSR construction on the tertiary industry agglomeration and provides an important reference for the synergy between HSR construction planning and tertiary industry clustering.