Background: Cancer is a complex systemic disease. As a key component of traditional Chinese medicine, acupuncture is a clinically proven medical treatment for many diseases, and it also has preventative effects as it balances the body, allowing it to self-regulate. For cancer patients, acupuncture is widely used as complementary therapy to boost the immune system and reduce the side effects of radiotherapy and chemotherapy. However, few studies have determined how acupuncture against cancer, especially in regulating the intestinal flora of the tumor-burdened mice. Methods: We treated osteosarcoma tumor-burdened mice by using needling on different acupoints and acupoints combination, thereafter determined the effects of acupuncture on tumor growth by using imaging technology in vitro. In addition, intestinal bacteria were analyzed for further understanding the holistic and systemic treatment effects of acupuncture in osteosarcoma tumor-burdened mice. Results: Acupuncture treatment can delay tumor growth and changes of intestinal bacteria in osteosarcoma tumor-burdened mice. In detail, the loss of body weight and the development of tumor volume of mice have been postposed by needling specific acupoints. In addition, acupuncture treatment has delayed the changes of the relative abundance of Bacteroidetes, Firmicutes and Candidatus Saccharibacteria at the phylum level. Moreover, the relative abundance of many bacteria (e.g., Catabacter, Acetatifactor and Aestuariispira) has been regulated by using acupuncture treatment, and the trend of structural changes of these bacteria at the genus level has also been postposed compared to that of the tumor-burdened mice model group. Conclusion: Our results suggest that acupuncture may provide a systemic treatment for cancer. Our findings encourage new and extensive research into the effects of acupuncture on changes of the intestinal microbiome associated with the development of cancer.