The opening of high-speed railways will help enterprises break the limitations of time and space, promote the free flow of capital factors between regions, and play an important role in smoothing domestic circulation. Existing empirical analysis has begun to pay attention to the significant promotion of capital flow by the opening of high-speed railways, but few studies have examined the impact and internal mechanism of high-speed railway opening on capital flow from the perspective of cross-regional investment by enterprises. Based on the cross-regional investment data of China’s A-share listed companies from 2006 to 2019, this paper examines the impact of high-speed railway opening on cross-regional investment of enterprises and explores its internal mechanism. The research results show that the opening of high-speed railways significantly improves the cross-regional investment level of enterprises, indicating that good transportation infrastructure can help smooth the domestic circulation; the promotion effect of high-speed railway opening on cross-regional investment of enterprises is stronger in the tertiary industry, technology-intensive industry enterprises, as well as enterprises located in “new infrastructure” developed areas, high-grade cities, and cities within the same urban agglomeration, so differentiated strategies should be adopted to support and encourage enterprises to seize the opportunity of high-speed railway opening and improve the level of cross-regional investment; the opening of high-speed railways promotes two types of cross-regional investment, namely “market-seeking” and “technology acquisition type”, so it is necessary to focus on breaking down interregional market segmentation and technical barriers, and fully release the positive role of high-speed railway in optimizing the domestic industrial structure; in addition, the optimal radius of the promotion effect of the opening of high-speed railway on cross-regional investment of enterprises is 600~900 kilometers, so it is possible to further strengthen the construction of new infrastructure in the area with high-speed railways, and promote cross-regional investment of enterprises through the linkage of high-speed railway and other new infrastructure. Compared with previous studies, this paper discusses the microeconomic effect of high-speed railway opening from the perspective of enterprise cross-regional investment, analyzes the mechanism of enterprise cross-regional investment, and finds that there is an optimal radius for the promotion effect of high-speed rail on enterprise cross-regional investment, expanding and deepening the research on the promotion effect of high-speed rail on enterprise cross-regional investment. At the same time, from the perspective of industry and regional heterogeneity, the heterogeneity of the promotion effect of high-speed railway opening on cross-regional investment of enterprises is analyzed, which is helpful to deeply understand the important role of good transportation infrastructure in smoothing domestic circulation. Based on the research conclusions of this paper, it is suggested that the government should accelerate the improvement of high-speed railway network construction, and take active measures to release the driving role of high-speed railway in optimizing the investment layout of enterprises, and enterprises should seize the opportunity of high-speed railway opening, enhance the endogenous driving force of investment, actively participate in cross-regional investment in the domestic cycle, and then efficiently play the role of high-speed railway in promoting cross-regional investment of enterprises.