The relationship between the leadership behavior of a chief executive officer (CEO) and start-up enterprise performance is key to effecting the survival and development of start-ups in the era of the internet economy. Currently, most studies on this topic focus more on the role of leadership effectiveness but rarely explore the CEO’s humility in leadership and the mechanism of its behavior affecting enterprise performance and its sustainable development. Based on leadership theory and upper-echelon theory, we build a research model of CEO’s humble leadership behavior, top management team’s (TMT’s) transactive memory system, and start-up enterprise performance, as well as the moderating roles exerted by strategic flexibility. Further, to validate the hypothesis, 400 valid questionnaires are obtained. Based on those data, the empirical results show humility, as a virtue, not only can significantly and positively improve start-up firm performance but also can promote the firm’s sustainable development in the long run by providing a trustworthiness climate for TMT members. Moreover, TMT’s transactive memory systems play a partial mediating role in the relationship between CEO’s humble leadership behavior and start-up enterprise performance. Meanwhile, strategic flexibility significantly and positively moderates the relationship between CEO’s humble leadership behavior and startup entrepreneurial performance. Finally, the theoretical and practical implications are discussed, and directions for future research are proposed.