Recent studies have shown that flagella may modulate physiological processes by sensing environmental changes in temperature and moisture. When the z66+ strain of Salmonella enterica serovar Typhi (S. Typhi) was exposed to an antiserum against the z66 flagellar antigen, the fljBA operon was deleted from a linear plasmid, leading to the unidirectional flagellar phase variation from FljB to FliC. We hypothesized that flagella may serve as a sensor that responded to the antiserum by altering gene expression and triggering the unidirectional flagellar phase variation. To test this hypothesis, Salmonella genomic DNA microarrays were used to determine the gene expression profile of the z66+ wild-type strain of S. Typhi treated with the anti-z66 antiserum for 30 min. The results showed that expression levels of 187 genes were altered by more than threefold compared with the same strain treated with control serum. The microarray expression patterns of representative genes were validated by reverse transcriptase-PCR. Importantly, no significant changes in gene expression were observed in the fljB:z66 deletion mutant that was similarly treated with the anti-z66 antiserum. To the best of our knowledge, this is the first study to show the global transcriptional response of Salmonella to antiflagellin antiserum.