β-Lactam antibiotics have cerebral and peripheral adverse effects. Multidrug resistance-associated protein 4 (MRP4) has been reported to transport several β-lactam antibiotics, and its expression at the blood-brain barrier also serves to limit their distribution to the brain. Therefore, the purpose of this study was to clarify the structure-activity relationship of MRP4-mediated transport of β-lactam antibiotics using MRP4-expressing Sf9 membrane vesicles. The transport activity was evaluated as MRP4-mediated transport per MRP4 protein [nL/(min·fmol MRP4 protein)] based on measurement of MRP4 protein expression by means of liquid chromatography-tandem mass spectrometry. Cefotiam showed the greatest MRP4-mediated transport activity [8.90 nL/(min·fmol MRP4 protein)] among the β-lactam antibiotics examined in this study. Measurements of differential transport activity of MRP4 for various β-lactam antibiotics indicated that (i) cephalosporins were transported via MRP4 at a greater rate than were penams, β-lactamase inhibitors, penems, or monobactams; (ii) MRP4-mediated transport activity of anionic cephalosporins was greater than that of zwitterionic cephalosporins; and (iii) higher-molecular-weight anionic β-lactam antibiotics showed greater MRP4-mediated transport activity than lower-molecular-weight ones, whereas zwitterionic β-lactam antibiotics did not show molecular weight dependency of MRP4-mediated transport. These quantitative data should prove useful for understanding MRP-related adverse effects of β-lactam antibiotics and their derivatives.