Objective To increase the resistance of ingested bacteria to multiple environmental stresses, the role of transglutaminase in Lactococcus lactis and possible mechanisms of action were explored. Results L. lactis grown with transglutaminase exhibited significantly higher resistance to bile salts, stimulated gastric juice, antibiotics, NaCl, and cold stress compared to the control (cultured without transglutaminase), with no negative influence on cell growth. Transmission electron microscopy revealed that the cell walls of L. lactis cultured with 9 U transglutaminase/ml were approx. 1.9-times thicker than the control. Further analysis demonstrated that the multi-resistant phenotype was strain-specific; that is, it occurred in bacteria with the presence of glutamine and lysine in the peptidoglycan. Conclusion Supplementation of culture media with transglutaminase is an effective, simple, and inexpensive strategy to protect specific ingested bacteria against multiple environmental challenges.