| Summary: | <i>Lactobacillus iners</i> is the most prevalent bacterial species in the human vaginal microbiome, and there have been few reports of its Gram-negative stain appearances despite the fact that the genus <i>Lactobacillus </i>is universally described as Gram-positive. Here, using transmission electron microscopy, we reveal that the thinness of the cell wall (17.39 ± 0.8 nm) gives the Gram-negative stain appearance, which can lead to over-diagnosis of bacterial vaginosis. Moreover, comparative genome analysis identified four genes commonly absent in <i>L.</i> <i>iners</i> genomes that might contribute to this phenotypic difference. We suggest that, along with the several niche-specific attributes identified, this unique feature may contribute to the species’ distinguished capability to thrive as the predominant species in the fluctuating vaginal environment as well.
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