Nicotine use is highly prevalent and brings a huge burden on individuals, society, health-care systems and economic development. The existing mechanisms underlying nicotine’ actions can’t illuminate all basic and clinical problems thoroughly. Transfer RNA-derived small RNAs (tsRNAs) is a novel class of small non-coding RNAs (sncRNAs), possessing potential regulatory functions in various diseases. However, the roles of tsRNAs in nicotine exposure have not been determined yet. In this study, firstly we established nicotine exposure model by subcutaneously injecting (sc.) with 0.5mg/kg of nicotine twice daily for 14 consecutive days, and conducted some behavioral observations (the pain threshold and body weight gains). Secondly, we identified the differentially expressed profiles of tsRNAs in rat hippocampus on saline or nicotine delivery conditions by using ncRNA-Seq, and then predicted the promising functions of the putative genes of the tsRNAs by bioinformatic method. The results shown that there were 26 differentially expressed tsRNAs (7 up-regulated and 19 down-regulated tsRNAs) (Fold change > 1.5; P < 0.05), of which the tRF-5 was the most common type. Eight tsRNAs were selected to validate the sequencing result by RT-qPCR. Then, based on the sequencing and RT-qPCR data, five candidate tsRNAs (tRF-1-T28-His-GTG-1, tRF-5c-Glu-CTC-1, tRF-5c-Glu-CTC-3, tRF-5c-Gly-GCC-2-M2, tRF-5c-Glu-TTC-4) were finally selected for further bioinformatic analysis. The GO and KEGG pathway enrichment analysis suggested that the five candidate tsRNAs might play regulatory roles through the cholinergic synapse pathways, dopaminergic synapse pathways, etc. In conclusion, our results indicated that tsRNAs were dysregulated in the rat hippocampus after nicotine exposure, and among them, tRF-5c-Glu-CTC-1 was the most promising one, which might lay a novel foundation for further research into nicotine’ actions.