Leukemia is a malignant tissue-forming disease, which induces the overproduction of large numbers of immature blood cells entering the peripheral blood. It is well documented that inflammation plays a crucial role in the expansion of leukemia. Daphnetin has confirmed anti-inflammatory effects against various diseases. In this experimental study, we evaluated the anti-leukemia and anti-inflammatory effect of daphnetin against benzene-induced leukemia in rats and explored the underlying mechanism. Benzene was used for inducing leukemia in experimental rats. The rats were divided into different groups and the body weight, hematological parameters, bone marrow cells, cytokines, and inflammatory mediators were estimated. Reverse transcription polymerase chain reaction (RT-PCR) was used for estimating the messenger RNA (mRNA) expression of sphingosine-1-phosphate receptor-1. Daphnetin-treated rats showed upregulation of body weight compared to other groups. Moreover, Daphnetin reduced blasts in leukemic rats. It also altered hematological parameters such as red blood cells, white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, monocytes, and basophils, respectively. Daphnetin-treated rats showed a reduction of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1 beta (IL-1 beta), IL-2, IL-6, and inflammatory mediators including nuclear factor-kappa B. RT-PCR showed upregulated mRNA expression of sphingosine-1-phosphate receptor-1 of daphnetin-treated group rats compared to other groups. The current study showed that the anti-inflammatory effect of daphnetin against the benzene-induced leukemia via alteration of cytokines.