The purpose of this study is to investigate the effects of 2.4 GHz Wi-Fi exposure, which is continuously used in the internet connection by mobile phones, computers and other wireless equipment, on microRNA and membrane and depot fatty acid composition of brain cells. Sixteen Wistar Albino rats were divided equally into two groups such as sham and exposure. The rats in the experimental group (n = 8) were exposed to 2.4 GHz RFR emitted from a Wi-Fi generator for 24 h/ day for one year. The animals in the control group (n = 8) were kept under the same conditions as the experimental group, but the Wi-Fi generator was turned off. At the end of the study, rats were sacrificed and brains were removed to analyze miRNA expression and membrane and depot fatty acids of brain cells. We analyzed the situation of ten different miRNA expressions and nineteen fatty acid patterns in this study. We observed that long-term and excessive exposure of 2.4 GHz Wi-Fi radiation increased rno-miR-181a-5p, phosphatidylserine (PS) and triacylglycerol (TAG) in the brain. In conclusion, 2.4 GHz Wi-Fi exposure has the potential to alter rno-miR-181a-5p expression and the fatty acid percentage of some membrane lipids such as phospholipid (PL), phosphatidylserine (PS) and triacylglycerol (TAG), which are depot fats in the brain. However, the uncontrolled use of RFRs, whose use and diversity have reached incredible levels with each passing day and which are increasing in the future, may be paving the way for many diseases that we cannot connect with today.