The birch pollen allergen Bet v 1 is believed to be the main sensitizer among PR-10 allergens. Recent data have shown that some other PR-10 allergens also display sensitization activities, and Bet v 1-based immunotherapy is not effective for blocking allergic reactions to PR-10 proteins with low similarities to Bet v 1. Here, we investigated the sensitization potential of the major soybean allergen Gly m 4 and its cross-reactivity with Bet v 1. We demonstrated that Gly m 4 bound cholesterol and bile acids, including deoxycholic acid (DCA). Using qPCR, we showed that Gly m 4 induced the expression of genes encoding alarmins TSLP and IL-33 in intestinal-like Caco-2 cells; however, its fragments resulting from digestion by gastroduodenal enzymes or the DCA-bound Gly m 4 caused more pronounced gene upregulation. Using competitive ELISA, we demonstrated the low cross-reactivity of anti-Gly m 4 and anti-Bet v 1 IgG, raised in laboratory animals. Using mice allergy models with sensitization to birch or soybean allergens, we also showed a low cross-reactivity of Gly m 4- and Bet v 1-specific IgE, IgG1 and IgG2a. Thus, our findings support an assumption of the intrinsic sensitization capacity of Gly m 4 and the existence of Gly m 4-specific antibodies in sera of allergic patients.