MicroRNAs (miRNA) are small non-coding RNAs that are key negative regulators of gene expression. Their roles include shaping the gene expression landscape during and after brain development by defining and maintaining levels of proteins that generate the distinct morphological and functional properties of neurons and other brain cell types. HT22, N2A, and SH-SY5Y are common immortalized neuronal cell lines that offer simple, less expensive, and time-saving options for in vitro modelling to evaluate miRNA functions. The extent to which these lines reflect primary neurons remains, however, unclear. Here, we benchmarked the miRNA profiles of cultured mouse hippocampal neurons against Argonaute-loaded miRNAs in the adult mouse hippocampus and miRNA data from the hippocampus of patients with drug-resistant temporal lobe epilepsy. We then compared the miRNA expression landscape in HT22, N2A and SH-SY5Y against mouse hippocampal primary cell cultures. We profiled over 700 miRNAs across the lines and detected 310 miRNAs in all four cell types. This included detection of neuron-enriched miRNAs such as miR-124 and miR-128, although the cell lines typically displayed lower levels of these than in primary neurons and reference adult hippocampal tissue. The miRNA profile in the HT22 cell line showed the highest correlation to the mouse primary neuronal cultures. Together, this study provides a dataset on basal miRNA expression across commonly used cell lines for neuroscience research and evidence for both conserved and distinct profiles that should be used to inform decisions on cell lines for modelling brain and miRNA research.