The dysregulation of microRNAs (miRNAs) is a crucial molecular signature of disease development. The potential implication of miRNAs in neurofibromatosis type 1 (NF1) remains poorly investigated. The expression levels of miR-27a-3p, miR-27b-3p, and neurofibromin 1 (NF1) were detected by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. The functional roles of miR-27a-3p and miR-27b-3p in NF1 were explored by CCK8 (Cell Counting Kit-8), 5-ethynyl-2'-deoxyuridine (EdU), terminal deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL), and transwell assays. Luciferase reporter, RNA pull-down, and RNA binding protein immunoprecipitation (RIP) assays were employed to study the probable target relationship between miRNA and messenger RNA (mRNA). MiR-27b-3p and miR-27a-3p were upregulated in dermal and plexiform human Schwann cells (HSC) from NF1 neurofibromas as well as cell lines of malignant peripheral nerve sheath tumors (MPNSTs). MiR-27a-3p/miR-27b-3p mimics promoted the proliferative, migratory, and invasive ability of dermal HSC and MPNST cell ST88-14, while inhibiting the apoptotic capacity. MiR-27a-3p/miR-27b-3p inhibitors elicited the opposite impacts on the above cellular behaviors in dermal HSC and ST88-14. Intriguingly, NF1 was revealed to be the target of both miR-27a-3p and miR-27b-3p, and was negatively modulated by them. MiR-27a-3p/miR-27b-3p upregulation suppressed the expression of NF1 in dermal HSC and ST88-14. Furthermore, NF1 depletion counterbalanced the functional alteration induced by miR-27a-3p/miR-27b-3p inhibition. Our study suggests that both miR-27b-3p and miR-27a-3p are involved in upstream molecular activity responsible for the depletion of NF1, representing promising targets for therapeutic application in NF1.