Cancer is the leading cause of death in both humans and companion animals. Canine mammary tumor is an important disease with a high incidence and metastasis rate, and its poor prognosis remains a serious clinical challenge. C6 ceramide is a short-chain sphingolipid metabolite with powerful potential as a tumor suppressor. However, the specific impact of C6 ceramide on canine mammary cancer remains unclear. However, the effects of C6 ceramide in canine mammary cancer are still unclear. Therefore, we investigated the role of C6 ceramide in the progress of canine mammary cancer and explored its potential mechanism. C6 ceramide inhibited cell growth by regulating the cell cycle without involving apoptosis. Additionally, C6 ceramide inhibited the migration and invasion of CHMp cells. In vivo, C6 ceramide decreased tumor growth and metastasis in the lungs without side effects. Further investigation found that the knockdown of EGR3 expression led to a noticeable increase in proliferation and migration by upregulating the expressions of pJAK1 and pSTAT3, thus activating the JAK1/STAT3 signaling pathway. In conclusion, C6 ceramide inhibits canine mammary cancer growth and metastasis by targeting EGR3 through the regulation of the JAK1/STAT3 signaling pathway. This study implicates the mechanisms underlying the anti-tumor activity of C6 ceramide and demonstrates the potential of EGR3 as a novel target for treating canine mammary cancer.