© 2018 Elsevier B.V. To investigate the relationship between intracellular acidification and inactivation of Saccharomyces pastorianus by a two-stage system with pressurized carbon dioxide microbubbles (two-stage MBCO2), the survivors and intracellular pH (pHin) of S. pastorianus suspended in buffers at various pHs after the two-stage MBCO2 treatment were measured. The inactivation efficiency of two-stage MBCO2 on S. pastorianus increased with lowering the initial buffer pH, although the pHin of S. pastorianus in pH 3 buffer was decreased by heating to 45 °C, regardless of the use of MBCO2. Conversely, the number of surviving S. pastorianus cells in pH 4 and 5 buffers was decreased by the two-stage MBCO2 without the pHin-lowering. Furthermore, the inactivation efficiency of two-stage MBCO2 on S. pastorianus increased with increasing pressure in the mixing vessel, although the pHin-lowering was caused by heating to 45 °C for all of the pressures tested, regardless of the use of MBCO2. Therefore, intracellular acidification of S. pastorianus by two-stage MBCO2 was due to the cell penetration of extracellular H+ associated with the temperature increase, and the inactivation was suggested to be induced by the combined factors of increasing temperature, pHin- and pHex-lowering and high dissolved CO2 concentration.