Learning and memory are fundamental processes that are disrupted in many neurological disorders including Alzheimer’s disease and epilepsy. The hippocampus plays an integral role in these functions, and modulation of synaptic transmission mediated by γ‐aminobutyric acid (GABA) type‐A receptors (GABA_ARs) impacts hippocampus‐dependent learning and memory. The protein diazepam binding inhibitor (DBI) differentially modulates GABA_ARs in various brain regions, including hippocampus, and changes in DBI levels may be linked to altered learning and memory. The effects of genetic loss of DBI signaling on these processes, however, have not been determined. In these studies, we examined male and female constitutive DBI knockout mice and wild‐type littermates to investigate the role of DBI signaling in modulating multiple forms of hippocampus‐dependent spatial learning and memory. DBI knockout mice did not show impaired discrimination of objects in familiar and novel locations in an object location memory test, but did exhibit reduced time spent exploring the objects. Multiple parameters of Barnes maze performance, testing the capability to utilize spatial reference cues, were disrupted in DBI knockout mice. Furthermore, whereas most wild‐type mice adopted a direct search strategy upon learning the location of the target hole, knockout mice showed higher rates of using an inefficient random strategy. In addition, DBI knockout mice displayed typical levels of contextual fear conditioning, but lacked a sex difference observed in wild‐type mice. Together, these data suggest that DBI selectively influences certain forms of spatial learning and memory, indicating novel roles for DBI signaling in modulating hippocampus‐dependent behavior in a task‐specific manner.