This study presents a protocol for the establishment of Prosopis laevigata cell suspension culture as a strategy to obtain an in vitro mesquite gum productive cell line. The callus used for this purpose was obtained with hypocotyls from 15-day-old plantlets, placed on Murashige–Skoog medium with two different plant growth regulators (PGRs), 2,4,5-trichlorophenoxy acetic acid (2,4,5-T; 5.0 µM) and kinetin (KIN; 5.0 µM). With this PGRs treatment, after four subcultures (30 days each) an exuded gum-like substance was observed on the callus surface. The growth kinetics of the cell suspension culture showed a specific cell growth rate (µ) of 0.14 d⁻¹ and doubling time (td) of 6.6 days, respectively. The gum-like substance from callus culture and the broth from cell suspension culture were subjected to chemical analysis and compared with the mesquite gum exuded from wild trees. Both, gum-like substance from callus culture and the broth from cell suspension culture showed the presence of Arabinogalactanproteins, and their polysaccharide fraction presented the same monosaccharides as those isolated from mesquite gum. In addition, the emulsifying properties of gum-like substance from callus culture and the broth from cell suspension culture were compared to those of mesquite gum and all three samples exhibited similar emulsifying capacity and emulsification stability.