The aim of the present study was to determine whether there is the mechanistic connection between antibacterial-dependent gut microbiota disturbance and anxiety. First, exposure of mice to ampicillin caused anxiety and colitis and increased the population of Proteobacteria, particularly Klebsiella oxytoca, in gut microbiota and fecal and blood lipopolysaccharide levels, while decreasing lactobacilli population including Lactobacillus reuteri. Next, treatments with fecal microbiota of ampicillin-treated mouse (FAP), K. oxytoca, or lipopolysaccharide isolated from K. oxytoca (KL) induced anxiety and colitis in mice and increased blood corticosterone, IL-6, and lipopolysaccharide levels. Moreover, these treatments also increased the recruitment of microglia (Iba1+), monocytes (CD11b+/CD45+), and dendritic cells (CD11b+/CD11c+) to the hippocampus, as well as the population of apoptotic neuron cells (caspase-3+/NeuN+) in the brain. Furthermore, ampicillin, K. oxytoca, and KL induced NF-κB activation and IL-1β and TNF-α expression in the colon and brain as well as increased gut membrane permeability. Finally, oral administration of L. reuteri alleviated ampicillin-induced anxiety and colitis. These results suggest that ampicillin exposure can cause anxiety through neuro-inflammation which can be induced by monocyte/macrophage-activated gastrointestinal inflammation and elevated Proteobacteria population including K. oxytoca, while treatment with lactobacilli suppresses it.