Remarkable sex difference has been observed in emotional processing including anxiety. The hippocampus, its ventral pole in particular, modulates anxiety-like behavior in rodents. However, most researches have been performed in male animals only, leaving hippocampal modulation of anxiety in females poorly defined. In the present study, we showed that excitotoxic lesioning of the ventral hippocampus with ibotenic acid produced anxiolytic effects in three behavioral tests (novelty-suppressed feeding, marble burying, and elevated-plus maze) in male but not female C57BL/6 J mice. Locomotion in the open field remained similar after lesioning in either sex. More c-Fos-positive neurons were observed in the ventral hippocampus in male than in female mice after exploration in an elevated plus-maze, indicating stronger enrollment of this region in anxiety-like behavior in males. These results reveal significant biological sex difference in ventral hippocampal modulation on anxiety in mice and provide a new sight for anxiety modulation and hippocampal function.