Backgrounds: Gut microbiota is increasingly recognized as an important environmental factor that could influence the brain function and behaviors through the microbiota-gut-brain axis. Method: Here, we used the germ-free (GF) mice to explore the effect of gut microbiota on hippocampal microRNA (miRNA) and messenger RNAs (mRNAs) expression. Results: Behavioral tests showed that, compared to specific pathogen-free (SPF) mice, the GF mice displayed more center time, center distance and less latency to familiar food. Colonization of the GF mice with gut microbiota from SPF mice did not reverse these behaviors. However, 7 differentially expressed miRNAs and 139 mRNAs were significantly restored. Through microRNA Target Filter analysis, 4 of 7 restored miRNAs had 2232 target mRNAs. Among these target mRNAs, 21 target mRNAs levels were decreased. Further analysis showed that the most significant GO terms were metabolic process (GO: 0008152), binding (GO: 0005488) and cell part (GO: 0044464) for biological process, molecular function and cellular component, respectively, and the most significantly altered pathway was axon guidance (mmu04360). Conclusions: These findings indicated that colonization of gut microbiota to adolescent GF mice was not sufficient to reverse the behavioral alterations. Gut microbiota could significantly influence the expression levels of miRNAs and mRNAs in hippocampus. Our results could provide original and valuable data for researchers to further study the microbiota-gut-brain axis. (C) 2017 Elsevier B.V. All rights reserved.