The primary quinone electron acceptor QA is a key component in the electron transfer regulation in photosystem II (PSII), and hence accurate estimation of its redox potential, E m(QA –/QA), is crucial in understanding the regulatory mechanism. Although fluorescence detection has been extensively used for monitoring the redox state of QA, it was recently suggested that this method tends to provide a higher E m(QA –/QA) estimate depending on the sample status due to the effect of measuring light Kato et al. (2019) Biochim. Biophys. Acta 1860, 148082. In this study, we applied the Fourier transform infrared (FTIR) spectroelectrochemistry, which uses non-reactive infrared light to monitor the redox state of QA, to investigate the effects of stromal- and lumenal-side perturbations on E m(QA –/QA) in PSII. It was shown that replacement of bicarbonate bound to the non-heme iron with formate upshifted E m(QA –/QA) by ∼55 mV, consistent with the previous fluorescence measurement. In contrast, an E m(QA –/QA) difference between binding of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and bromoxynil was found to be ∼30 mV, which is much smaller than the previous estimate, ∼100 mV, by the fluorescence method. This ∼30 mV difference was verified by the decay kinetics of the S2QA – recombination. On the lumenal side, Mn depletion hardly affected the E m(QA –/QA), confirming the previous FTIR result. However, removal of the extrinsic proteins by NaCl or CaCl2 wash downshifted the E m(QA –/QA) by 14–20 mV. These results suggest that electron flow through QA is regulated by changes both on the stromal and lumenal sides of PSII.