RNA sequencing (RNA-Seq), a revolutionary tool for transcriptome profiling, is becoming increasingly important for neuroscientists in studying the transcriptional landscape of the human brain. Studies using this next-generation sequencing technique have already revealed novel insights into the complexity of neurons in the human brain and pathogenesis of complex neurological diseases. In clinical neuroscience, RNA-Seq provides exciting opportunities for improving diagnosis and treatment of neurological diseases by facilitating the development of pharmacotherapies able to modulate gene expression. Furthermore, integrative whole genome sequencing and transcriptome sequencing can provide additional information for the functional role of mutated genes, prioritization of variants, and intron/exon splicing. This review describes the current state of RNA-Seq studies in neuropsychiatric disorders using post-mortem human brains, a brief survey of best practices for experimental design and sequencing data analysis, and the challenges associated with its application in the human brain.