Intestinal microbiota and their metabolites are essential for maintaining intestinal health, regulating inflammatory responses, and enhancing the body’s immune function. An increasing number of studies have shown that the intestinal microbiota is tightly tied to tumorigenesis and intervention effects. Intermittent fasting (IF) is a method of cyclic dietary restriction that can improve energy metabolism, prolong lifespan, and reduce the progression of various diseases, including tumors. IF can affect the energy metabolism of tumor cells, inhibit tumor cell growth, improve the function of immune cells, and promote an anti-tumor immune response. Interestingly, recent research has further revealed that the intestinal microbiota can be impacted by IF, in particular by changes in microbial composition and metabolism. These findings suggest the complexity of the IF as a promising tumor intervention strategy, which merits further study to better understand and encourage the development of clinical tumor intervention strategies. In this review, we aimed to outline the characteristics of the intestinal microbiota and its mechanisms in different tumors. Of note, we summarized the impact of IF on intestinal microbiota and discussed its potential association with tumor suppressive effects. Finally, we proposed some key scientific issues that need to be addressed and envision relevant research prospects, which might provide a theoretical basis and be helpful for the application of IF and intestinal microbiota as new strategies for clinical interventions in the future.