Although the exact mechanism(s) by which estradiol (E2) enhances muscle growth in a number of species, including humans and cattle, is not known, E2 treatment has been shown to stimulate proliferation of cultured bovine satellite cells (BSCs). This is particularly significant because satellite cells are the source of nuclei needed to support postnatal muscle fiber hypertrophy and are thus crucial in determining the rate and extent of muscle growth. The objective of this study was to assess the role of estrogen receptor (ESR1) and the type 1 insulin-like growth factor receptor (IGFR1) in E2 stimulated proliferation of cultured BSCs. To accomplish this, we have used small interfering RNA (siRNA) to silence expression of ESR1 or IGFR1 and assessed the effects on E2-stimulated proliferation in BSC cultures. In BSCs treated with nonspecific siRNA, E2 significantly (P < 0.05) stimulates proliferation under conditions in which neither IGF-1 nor IGF-2 expression is increased; however, treatment of ESR1- or IGFR1-silenced cells with E2 does not significantly stimulate proliferation. These results indicate that both ESR1 and IGFR1 are required for E2 to stimulate proliferation in BSC cultures. The fact that this occurs under culture conditions in which neither IGF-1 nor IGF-2 mRNA expression is increased strongly suggests that E2 activates IGFR1 via a mechanism that does not involve increased IGF-1 or IGF-2 binding to the receptor.