Methylphenidate (MPH) is a central nervous system stimulant that is widely used to treat attention deficit hyperactivity disorder (ADHD) and has been shown to improve attention, cognitive function and behaviors in both patients and animal models of ADHD. Even among normal healthy people, MPH can facilitate the consolidation of memories and improve declarative memory. Using microarray techniques, we aimed to find new pharmacology profile of MPH. A Làt maze experiment showed that locomotor activity and non-selective attention were affected by 2 weeks of exposure to MPH. Then, we identified long non-coding RNA (lncRNA) signatures in the prefrontal cortex of rats; 461 up-regulated lncRNAs and 97 down-regulated lncRNAs were found in the MPH-exposed group compared with the control group using fold-change >1.5. GO and KEGG pathway analyses indicated biological functions related to the metabolism of neural chemical compounds and nerve cell development. Furthermore, we reported changes in uc.173+ related to the UBE2B gene, which may affect neurite outgrowth and axonal regeneration. At the same time, MRAK081997 associated with the DHFR gene may be involved in axon regeneration in the rodent central nervous system through DNA methylation. Our study showed distinct expression profiles of lncRNAs in the normal rat prefrontal cortex after exposure to MPH, offering information for further research of MPH and may suggesting a new therapeutic target for ADHD.