Jerusalem artichoke (Helianthus tuberosus L.) is an herbaceous perennial plant in Compositae with certain salt tolerance, but its salt-responsive mechanisms have not been clearly revealed. Suspension cell cultures act as a model system and can support a consistent and coordinated cellular response to salt stress. In the present study, a combination of physiological characteristics and tandem mass tags (TMT)-based proteomic approaches was conducted to investigate the salt response of suspension cell cultures of Jerusalem artichoke. NaCl stress significantly inhibited the proliferation of Jerusalem artichoke suspension cells and caused a decrease in relative cellular activity. In addition, the content of soluble sugar, proline, H2O2, and malondialdehyde was affected by salt stress in suspension cells. From the proteomic analysis, a total of 313 upregulated proteins and 365 downregulated proteins were identified after salt stress. Functional analysis showed that the differentially expressed proteins were involved in a variety of biological processes, including the division of cells, reconstruction of water balance components, scavenging of reactive oxygen species (ROS), biogenesis of ribosomes and proteins, and metabolism of carbohydrates. These findings suggest that salt stress affects protein synthesis and cell division and induces the expression of proteins important for the control of ROS production and metabolic homeostasis in Jerusalem artichoke cell culture.