Callus cultures initiated from red beet tubers were acclimated to 75 or 100 mM NaCl salinity by exposing them to gradually increasing NaCl concentrations. The acclimated callus lines displayed growth rates comparable to the control culture cultivated on the NaCl-free medium. Several antioxidant system components were analyzed to assess the role of the antioxidant defense in the acclimated callus’s ability to proliferate on salt-supplemented media. It was found that proline and ascorbate concentrations were increased in salt-acclimated callus lines with respect to the control line. On the other hand, glutathione concentration was unchanged in all tested callus lines. Total activities of the antioxidant enzymes, namely superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and class III peroxidase (POX, EC 1.11.1.7) were increased in salt-acclimated cultures. The enzymatic components of the antioxidant systems were upregulated in a coordinated manner during the initial phases of the culture cycle when the increase in callus fresh mass occurs.