The supply chain is the environment on which an enterprise relies for survival, profoundly influencing its resource acquisition capability and subsequently leading to significant differences in strategic decisions of enterprises. This paper takes the Ashare nonfinancial listed companies in China from 2009 to 2023 as the research object, exploring the impact of major customers as suppliers on an enterprise’s diversified operation from the dual perspectives of “resources support” and “resources constraint”. The research finds that major customers as suppliers significantly inhibit an enterprise’s diversified operation, presenting a “resources constraint” effect. Its main mechanism of action is resources locking and exacerbating financing constraints. Further analysis shows that major customers as suppliers mainly inhibit the degree of unrelated diversified operation of enterprises; the inhibitory effect of major customers as suppliers on an enterprise’s diversified operation is more significant when there are fewer redundant resources, and this inhibitory effect becomes stronger with the persistence of the relationship of major customers as suppliers. Economic consequence tests show that reducing the degree of corporate diversification can achieve the growth of enterprise value and enhance supply chain resilience. The above research conclusions not only enrich the literature related to major customers and suppliers and enterprise diversified operation but also provide reference experience for enterprises to choose an appropriate business strategy based on their own resources acquisition capabilities. They also provide new ideas for enterprises to better cope with supply chain risks.