Bacteria that are Gram-negative are not stained dark blue or violet by Gram staining, in contrast to Gram-positive bacteria. On most Gram-stain preparations, Gram-negative organisms will appear red or pink because they are counterstained.

The difference lies in the cell wall of the two types; in contrast to most Gram-positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide. The space between the layers of peptidoglican and the secondary cell membrane is called periplasmatic space.

Many species of Gram-negative bacteria are pathogenic. This pathogenic capability is usually associated with certain components of their cell walls, particularly the lipopolysaccharide (endotoxin) layer.

The proteobacteria are a major group of Gram-negative bacteria, including for instance Escherichia coli, Salmonella, and other Enterobacteriaceae, Pseudomonas, Moraxella , Helicobacter, Stenotrophomonas , Bdellovibrio, acetic acid bacteria, Legionella and a great many others. Other notable groups of Gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria.