The study aimed to construct a recombinant Lactobacillus casei expressing the nonstructural (NS) 1 protein of influenza A virus H5N1 on its cell wall. The NS1 gene was first amplified and fused to the pSGANC332 expression plasmid. The NS1 protein expression was carried out by Lact. casei strain C1. PCR screening and DNA sequencing confirmed the presence of recombinant pSG-NS1-ANC332 plasmid in Lact. casei. The plasmid was stably maintained (98·94 ± 1·65%) by the bacterium within the first 20 generations without selective pressure. The NS1 was expressed as a 49-kDa protein in association with the anchoring peptide. The yield was 1·325 ± 0·065 μg mg(-1) of bacterial cells. Lactobacillus casei expressing the NS1 on its cell wall was red-fluorescently stained, but the staining was not observed on Lact. casei carrying the empty pSGANC332. The results implied that Lact. casei strain C1 is a promising host for the expression of surface-bound NS1 protein using the pSGANC332 expression plasmid.SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated, for the first time, the expression of nonstructural 1 (NS1) protein of influenza A virus H5N1 on the cell wall of Lactobacillus casei using the pSGANC332 expression plasmid. Display of NS1 protein on the bacterial cell wall was evident under an immunofluorescence microscopic observation. Lactobacillus casei carrying the NS1 protein could be developed into a universal oral influenza vaccine since the NS1 is highly conserved among influenza viruses.