Uterine leiomyomas, also known as fibroids, are common and prevalent in women of reproductive age. In this study, the effect of Isoliquiritigenin (ISL), a licorice flavonoid, on the anti-proliferation of uterine leiomyoma was investigated. We found that the survival of uterine leiomyoma ELT3 cells and primary uterine smooth muscle (UtSMC) cells was reduced by treatment with ISL alone or with ISL plus estradiol (E2). Cell cycles were arrested through the reduction of G2/M- and S-phase populations in ELT3 and UtSMC cells, respectively. Furthermore, increased sub-G1 phase and nucleus condensation were observed in ELT3 cells but not in UtSMC cells. Co-treatment of ELT3 cells with E2 and ISL inhibited ERK1/2 activation, whereas p38 and c-Jun N-terminal kinase (JNK) activation was enhanced. Moreover, ISL-induced apoptosis and autophagy cell death in ELT3 cells were observed. Serum E2 and P4 levels were reduced in a E2-enhanced uterine myometrium hyperplasia mouse model by ISL treatment, which contributed to the downregulation of the expression of extracellular matrix (ECM) associated proteins and matrix metalloproteinase (MMPs). Taken together, these results showed that ISL exerted a higher effect on the inhibition of estrogen-induced uterine leiomyoma growth for both in vitro and in vivo ECM accumulation, demonstrating its potential as a new option for treatment of uterine leiomyoma.