Transportation infrastructure construction can effectively reduce commuting costs and transportation costs, which plays an important role in optimizing industrial layout and advancing high-quality economic development. Based on identifying the overall effects of intra-regional commuting costs and inter-regional transportation costs on industrial agglomeration, this paper analyzes the heterogeneity of cities and industries in terms of geographic location, urban cluster spatial structure and the way of industrial factor concentration, and explores their mechanisms in terms of innovation level, talent attraction, market potential and labor mobility. It is found that the reduction of intra-regional commuting cost helps to promote industrial agglomeration and significantly improves industrial agglomeration efficiency; while the reduction of inter-regional transportation cost has an inverted U-shaped relationship with industrial agglomeration scale and a positive U-shaped relationship with industrial agglomeration efficiency. That is to say, when the transportation cost is reduced to a critical value, although it will prompt the industry to spread outward, it can improve the agglomeration efficiency. These effects exhibit heterogeneity across cities and industries. At the city level, the reduction of commuting cost is more significant for the industrial agglomeration scale of central and western regions and multi-center city clusters, and there is a positive U-shaped relationship between the transportation cost and the agglomeration efficiency of central and western cities. At the industrial level, the reduction of commuting cost favors the agglomeration of information-intensive industries, but has no significant effect on their agglomeration efficiency, while knowledge-intensive industries are more likely to spread outward. The reduction of inter-regional transportation cost helps to improve the agglomeration efficiency of knowledge-intensive industries, but the effect on information-intensive industries is not obvious. Mechanism test results show that the reduction of intra-regional commuting costs attracts talents to agglomerate, thus enhancing industrial agglomeration scale and efficiency. The inverted U-shaped effect of inter-regional transportation cost reduction on agglomeration scale is mainly realized by boosting urban market potential, and the positive U-shaped effect on agglomeration efficiency is realized by enhancing labor force mobility. It is further found that the impact of lower inter-regional transportation costs on the industrial agglomeration effect in neighboring areas does not show a clear U-shaped characteristic. The article concludes that it is necessary to promote the construction of transportation infrastructure according to local conditions, optimize industrial spatial distribution, and improve resource allocation efficiency. The eastern region and big cities can rely on the existing transportation network to promote the orderly transfer of industries; the central and western regions and small and medium-sized cities should strengthen the internal and external transportation connections and actively undertake industrial transfers; single-center city clusters need to reasonably guide the investment to avoid duplicated construction; multi-center city clusters should improve the nodal transportation system and promote regional synergistic development. In view of different industrial characteristics, it is necessary to focus on reducing commuting costs in promoting information-intensive industrial agglomeration, and strengthening inter-city transportation links in promoting knowledge-intensive industrial agglomeration. Finally, through differentiated transportation infrastructure construction and industrial layout optimization, China’s coordinated regional development will be promoted to a higher level, and sustained momentum will be injected into China’s high-quality economic development.