The significant antihypercholesterolemic effect of the undigested high molecular fraction (HMF) of soybean protein is known in rats, but such an effect has not been shown in humans. The present two experiments were designed to elucidate it in humans. Subjects were female university students who had relatively high serum cholesterol levels for their age. In Experiment 1, subjects took 8% of their total energy from casein, soybean protein isolate (SPI), or HMF daily for 14 days. Five basic menus and snacks were cycled. Energy intakes and daily activities were kept constant and body weight was maintained. The HMF group showed decreased low-density lipoprotein cholesterol (LDL-C) as compared to other groups. In Experiment 2, subjects took 4% of total energy from casein or HMF daily for a menstruation period. Five basic menus and snacks which contained two egg yolks (about 500 mg cholesterol) were cycled. Energy intakes and daily activities were kept constant and body weight was maintained. A decrease in LDL-C and an increase in high-density lipoprotein cholesterol (HDL-C) were observed in the HMF group as compared to the casein group. Fecal acidic steroid excretion was greater in the HMF group than in the casein group (p < 0.05). The results confirmed that HMF increases fecal steroid excretion and reduces serum cholesterol levels in humans.