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The CS21 pilus produced by enterotoxigenic <i>Escherichia coli</i> (ETEC) is involved in adherence to HT-29 intestinal cells. The CS21 pilus assembles proteins encoded by 14 genes clustered into the <i>lng</i> operon. Aim. This study aimed to determine whether <i>E</i>. <i>coli</i> BL21 (ECBL) transformed with the <i>lng</i> operon lacking the <i>lngA</i> gene (pE9034AΔ<i>lngA</i>) and complemented <i>in trans</i> with <i>lngA</i> variants of ETEC clinical strains, as well as point substitutions, exhibited modified adherence to HT-29 cells. Methods. A kanamycin cassette was used to replace the <i>lngA</i> gene in the <i>lng</i> operon of the E9034A strain, and the construct was transformed into the ECBL strain. The pJET1.2 vector carrying <i>lngA</i> genes with allelic variants was transformed into ECBLpE9034AΔ<i>lngA</i> (ECBLΔ<i>lngA</i>). The point substitutions were performed in the pJET<i>lngA</i>_FMU073332 vector. Results. Bioinformatic alignment analysis of the LngA proteins showed hypervariable regions and clustered the clinical ETEC strains into three groups. Variations in amino acid residues affect the adherence percentages of recombinant ECBL strains with <i>lngA</i> variants and site-specific mutations with HT-29 cells. Conclusion. In this study, ECBL carrying the <i>lng</i> operon harboring <i>lngA</i> variants of six clinical ETEC strains, as well as point substitutions, exerted an effect on the adherence of ECBL to HT-29 cells, thereby confirming the importance of the CS21 pilus in adherence.