Domestic chickens are an established model organism for studies on learning and memory. Commonly, the chicks are used as subjects in several different learning tests, including one trial learning tests. However, for adult laying hens no such one trial learning tests have been established. In particular, there is no test established which focuses on the role of the hippocampus, a brain region, which is often critically involved in learning and memory consolidation. In this study we investigated the inhibitory effects of intra-hippocampal D-AP5 injections on a specific one trial passive avoidance learning test in adult laying hens (Gallus gallus domesticus). We used a step down avoidance learning paradigm (SDA) which is frequently used in rodents. Intra-hippocampal injections of D-AP5 impaired the learning abilities of adult laying hens compared to sham-injected control subjects. Thus, the experiments revealed that the hippocampus is critically involved in learning the inhibitory SDA task. Our results further indicate that the step down avoidance paradigm is suitable to examine learning and memory processes in adult laying hens.