Lipoxin A4 (LXA4), an arachidonic acid-derived anti-inflammatory lipid mediator, shows anti-tumor potential by regulating tumor immune microenvironments. However, the underlying molecular and cellular basis of this function remains unclear. IL-10-producing B (Breg) cells display tumor-promoting effects by negatively regulating anti-tumor immunity. Here we show that LXA4 inhibits tumor growth by suppressing the generation of Breg cells in tumor-bearing mice. The administration of LXA4 inhibited the induction of Breg cells. Breg cell deficiency, in turn, resulted in LXA4 losing its anti-tumor properties. Intriguingly, regulatory T (Treg) cells also had a role in this process. Targeting Breg cells by LXA4 decreased the number of Treg cells in draining lymph nodes and tumor tissues as well as enhanced cytotoxic T cell activities. In addition, we further demonstrated that LXA4 inhibited Breg cells through its dephosphorylating STAT3 and ERK. These findings unveil a new anti-tumor mechanism underlying LXA4 targeting Breg cells with potential clinical applications.