Acid resistance is critical for the survival of Escherichia coli O157:H7 in acidic environments. The representative space environment microgravity is known to have a great impact on bacteria, but the acid stress response of E. coli O157:H7 under microgravity conditions remains unclear. Here, we show that the acid resistance of sbacteria is altered by the upregulation of related resistance systems. All tested E. coli O157:H7 strains (ATCC 35150, 43889, 43890, and 43895) survived better in acidified Luria-Bertani medium (pH 3.5) under low-shear modeled microgravity (LSMMG) than under normal gravity (NG, counterpart condition). For example, after 72 h of cultivation under acidic conditions, bacterial populations in the LSMMG cultures reached 5.2–6.7 log CFU/ml, while those in the NG cultures reached 2.4–5.6 log CFU/ml. Our transcriptomic analysis studies on E. coli O157:H7 under LSMMG conditions also provided supportive data of the increase in the acid stress response, with a 2.18 to 3.44 log2 fold change in the acid resistance system 1 (rpoS) and 2 (gad) and chaperone related genes (hdeA and hdeB). Comparing D-values before and after acid shock at pH 3.5, the increase in thermal cross-protection power was more remarkable in the LSMMG cultures than in the NG cultures. In the case of E. coli O157:H7 ATCC 35150, the D-values in the LSMMG and NG cultures at 55 °C after acid shock increased by 17.1 and 10.8 min, respectively, compared to the control. Our findings illustrated that simulated microgravity impacts the acid resistance of E. coli O157:H7 as well as the acquisition of thermal cross-protection power, suggesting that alterations in bacterial responses to the space environment could be a health threat.