PurposeWell-constructed transportation infrastructure may effectively decrease barriers to the flow of innovative human resources and inventive elements, accelerating enterprise innovation activities. This study will explore how HSR helps enterprises achieve ambidextrous innovation, including the mediating mechanism of absorbed slack resources, innovative talents, and the heterogeneous effects of management shareholding ratio and financing constraints.Design/methodology/approachBased on resource dependence theory and social network theory, this study employs a quasi-natural experiment of China’s high-speed railway and builds a multi-time point DID model to investigate its influence on enterprise ambidextrous innovation.FindingsResults suggest that the HSR positively influences both exploitative and exploratory innovation, and the influence is more substantial on exploitative innovation. Further analysis finds two influencing channels through which HSR influences enterprise ambidextrous innovation: providing redundant resources and attracting innovative talents. Heterogeneity analysis indicates that HSR has a more significant positive effect on exploratory innovation for enterprises with high management shareholding. In the low financing constraint group, the HSR opening has a more significant impact on ambidextrous innovation.Practical implicationsIn ambidextrous innovation, enterprises should rationalize the allocation of resources, attach importance to the innovative talent introduction, and choose differentiated paths based on intrinsic characteristics. Meanwhile, the government should actively improve the HSR routes and continuously improve the innovative environment.Originality/valueThis study enriches the theoretical research framework of HSR and ambidextrous innovation by identifying the channel mechanisms and boundary conditions through which HSR affects ambidextrous innovation and expands the consequences of HSR and the antecedents of ambidextrous.