As the responsible subject of environmental governance, the level of environmental investment of enterprises directly affects the sustainable development of country. And in the environmental protection investment decision of enterprise, the company governance structure plays a key role. As a special shareholding structure, multiple major shareholders may affect the environmental protection investment of enterprises. Taking China's A-share listed companies from 2009 to 2020 as a sample, the relationship between the equity structure of multiple major shareholders and the environmental protection investment of enterprises is examined. The research shows that the ownership structure of multiple major shareholders inhibit the scale of enterprise environmental protection investment, and the more multiple major shareholders and the higher the total shareholding ratio, the more unfavorable the enterprise environmental protection investment. Finally, the robustness test is carried out and the conclusion was still valid. The analysis of the relationship between the two is expected to provide some enlightenment for further research on the ownership structure of several major shareholders and corporate environmental investment.