Abstract: Strengthening enterprises' cross-regional cooperative innovations plays an important part in the construction of an innovative country. This paper collects the detailed cooperative patent application data of Chinese A-share listed companies from 2005 to 2019, and uses the difference-in-differences method to test the impacts of the opening of high-speed railway (HSR) on enterprises' cross-regional cooperative innovations. The study finds that the opening of HSR significantly improves the number of enterprises' cross-regional cooperative patent applications. This conclusion is also robust after the common trend test, placebo test, using the variable that whether a city opened the railway in 1945 as an instrument variable and different estimation methods. The mechanism test shows that the opening of HSR enhances enterprises' cross-city cooperative innovation mainly through the substitution effect of cross-city for intra-city cooperative innovation, technological upgrading effect, rather than the spatial competition effects of "zero-sum games" between regions. Further analysis reveals that the opening of HSR leads to the spatial distribution of enterprises' cooperative innovation partners moving to a longer distance, and has a greater role in promoting cross-city cooperative innovation of firms which are in non-high-tech industries and far away from the urban center. This paper provides new insights to promote enterprises' cross-regional cooperative innovation and build a regional coordinated national innovation system by the construction of HSR.