Exposure to radiofrequency (RF) electromagnetic fields (EMFs) has been linked to changes in neuronal electrophysiological properties and synaptic plasticity. Given that these types of changes could parallel alterations in gene expression, we sought to examine the effect of exposure to RF-EMFs on gene expression. We exposed rat primary hippocampal neurons (PHNs) to a single (one time) or multiple (three times) exposure to continuous wave RF-EMFs at two different doses (mean specific absorption rate (SAR) values of 0.57 W/kg or 5.91 W/kg) or were mock-exposed (sham). Changes in gene transcription in the RF-EMF exposed PHNs versus the sham counterparts were evaluated at 0, 4, and 24 h post-exposure via high-throughput ribonucleic acid (RNA) sequencing using Illumina HiSeq 2000. Significant differentially expressed genes (DEGs) were defined as having an adjusted p-value ≤ 0.05 and an absolute fold change ≥ 2.0 (twofold difference above or below the sham control). Using these criteria, we identified 20 genes displaying significant modulation due to RF-EMF exposure that were seen only with exposures at the higher dose (i.e., 5.91 W/kg), which also resulted in a temperature rise in the media up to 3.6 ± 0.6 °C. Several of the identified 20 DEGs play a role in the stress response mechanism. Interestingly, the expression of all 20 DEGs appeared to revert to baseline at 24 h after exposure, since their expression in the RF-EMF and sham samples was comparable. Therefore, our results suggest that RF-EMFs for the evaluated exposure parameters and conditions would not induce biologically relevant changes in gene expression.