Soy isoflavones, found in soybean and soybean products, have been reported to possess many physiological activities such as antioxidant activity, inhibition of cancer cell proliferation, reduction of cardiovascular risk, prevention of osteoporosis and alleviation of postmenopausal syndrome. In our previous study, soy isoflavone extract ISO-1 (containing 12 soy isoflavones) from soybean cake was demonstrated to prevent skin damage caused by UVB exposure. In this study, soy isoflavone extract from soybean cake was further purified and evaluated for the protective effects on UVB-induced damage. The results revealed that Fraction 3, which contains the aglycone group (daidzein, genistein and glycitein) and acetylglucoside group (acetyldaidzin, acetylgenistin and acetylglycitin) of soy isoflavones, could inhibit UVB-induced death of human keratinocytes and reduce the level of desquamation, transepidermal water loss (TEWL), erythema and epidermal thickness in mouse skin. Furthermore, topical application of Fraction 3 increased the activity of catalase and suppressed cyclooxygenase-2 (COX-2) and proliferating cell nuclear antigen (PCNA) expression in mice exposed to UVB. In addition, in comparison with ISO-1 and genistein, the Fraction 3 possessed much greater protective effects on both UVB-induced oxidative stress and keratinocyte death than other fractions. Therefore, the soy isoflavone extract Fraction 3 from soybean cake is a desirable anti-photoaging agent for skin care.