Pathogens that adapt to environmental stress can develop an increased tolerance to some physical or chemical antimicrobial treatments. The main objective of this study was to determine if acid adaptation increased the tolerance of Escherichia coli O157:H7 to high voltage atmospheric cold plasma (HVACP) in raw pineapple juice. Samples (10 ml) of juice were inoculated with non-acid-adapted (NAA) or acid adapted (AA) E. coli to obtain a viable count of ~ 7.00 log10 CFU/ml. The samples were exposed to HVACP (70 kV) for 1 to 7 min with inoculated non-HVACP-treated juice serving as control. Juice samples were analyzed for survivors at 0.1 h, and after 24 h of refrigeration (4 °C). Samples analyzed after 24 h exhibited significant decreases in viable NAA cells with sub-lethal injury detected in both NAA and AA survivors (P<0.05). No NAA survivor in juice exposed to HVACP for 5 or 7 min was detected after 24h. However, numbers of AA survivors were 3.33 and 3.09 log10 CFU/ml in juice treated for 5 and 7 min, respectively (P<0.05). These results indicate that acid adaptation increases the tolerance of E. coli to HVACP in pineapple juice. The potentially higher tolerance of AA E. coli O157:H7 to HVACP should be considered in developing safe juice processing parameters for this novel non-thermal technology.