This study was conducted to evaluate the estrogen activity of silkworm (Bombyx mori) pupaextracts and their fractions. Powdered samples of freeze-dried silkworm pupa were extracted atroom temperature (RT), 40ºC, 60ºC, 80ºC, and 100ºC in water (D.W), chloroform, ethylacetate, and methanol for 6h and then filtered (0.45 um). The extracts were then freeze-dried.The estrogenic activity of these extracts was then investigated by competition binding assaysusing estrogen receptor α (ERα) and ERβ, and by evaluating their effects on the proliferation ofthe human breast cancer cell line, MCF-7. Among the extracts evaluated, water extractsprepared at RT showed the highest binding affinity to ERα (IC50, 1.76 ug/ml) and ERβ (IC50,0.07 ug/ml). In addition, MCF-7 cells that were treated with 62.5 ug/ml of the RT extractshowed the greatest increase in proliferation (2-fold; 1291.79%) when compared to control cells(659.82%). Next, the water extract that was prepared at RT (sample 1) was dissolved in D.W.and further fractionated using a Dowex 50W - 8X (H+) column. The flow-through and washwere then pooled together and freeze-dried (sample 2). The bound materials were then elutedwith 20 mM NaCl, after which they were applied to a Dowex 1X2 - 200 (Cl-) column andwashed with D.W. to remove the sodium ions. The eluants were then freeze-dried (sample 3).Of these fractions, sample 2 showed the highest binding affinity to ERα (IC50, 1.44 ug/ml) andERβ (IC50, 1.18 ug/ml). In addition, MCF-7 cells that were treated with sample 2 (15.6 ug/ml)showed the largest increase in growth (1159.39%) when compared to control cells (525.26%).Taken together, these results suggest that the fraction of the RT water extract of silkworm pupareferred to as sample 2 may be useful as a phytoestrogen.