The membrane pore size and operating condition effects on the filtration flux, membrane fouling and solute rejections in cross-flow microfiltration of BSA/dextran binary suspension are studied. Two flat sheet microfiltration membranes with mean pore sizes of 0.1 and 0.025 μm are used as filter media in these experiments. The filtration flux increases with increasing cross-flow velocity and filtration pressure because of less membrane fouling or higher filtration driving force. The filtration resistance caused by membrane fouling is much higher than that caused by concentration polarization or virgin membrane. This impact is more significant under higher transmembrane pressures. The membrane fouling extent due to dextran adsorption is expressed as pore size reduction and fouled layer depth, which can be estimated theoretically based on hydrodynamic models. The dextran rejection decreases with increasing Reynolds number in the membrane pores and approaches constant when the Reynolds number exceeds a critical value. This is attributed to the increase in wall shear stress and molecular deformation in the membrane pores. A higher cross-flow velocity results in higher dextran rejection because of the sweeping effect occurring on the membrane surface. The BSA rejection under various operating conditions can be reasonably explained by the membrane sieving effect. The BSA rejection decreases linearly with increasing pore size in the fouled membrane with no obvious cross-flow velocity effect on BSA rejection found. The effects of the dextran concentration and Reynolds number in the membrane pores on the dextran adsorption layer thickness are also discussed.