Genistein and daidzein are known to have both beneficial and adverse effects on human health due to their many biological actions at the cellular level. Both isoflavones have been shown to inhibit GLUT-mediated glucose transport across the plasma membrane of mammalian cells. Since lysosomal membrane transport is essential for maintaining cellular homeostasis, the present study examined the effects of genistein and daidzein on glucose and sulphate transport in isolated rat liver lysosomes. Both genistein and daidzein significantly inhibited lysosomal glucose uptake. Genistein was a more potent glucose transport inhibitor than daidzein, with a half-maximum inhibitory concentration (IC50) of 45 μmol/l compared with 71 μmol/l for daidzein. Uptake kinetics of d-glucose showed a significant decrease in Vmax (control:genistein treat = 1489 (sem 91):507 (sem 76) pmol/unit of β-hexosaminidase per 15 s) without a change in Km. The presence of 50 μm-genistein in the medium also reduced glucose efflux from lysosomes preloaded with 100 mm-d-glucose. Genistein also inhibited lysosomal sulphate transport. Similar to its effects on glucose uptake kinetics, genistein treatment caused a significant decrease in sulphate uptake Vmax (control:genistein treat = 87 (sem 4):59 (sem 5) pmol/unit of β-hexosaminidase per 30 s), while the Km was not affected. The evidence provided by the present study suggests that the most likely mechanism of lysosomal glucose transport inhibition by genistein is via direct interaction between genistein and the transporter, rather than mediation by tyrosine kinase inactivation. Genistein likely has a similar mechanism of directly inhibiting sulphate transporter.