The effects of short-term social isolation during adulthood have not yet been fully established in rats behaviourally, and not at all transcriptomically in the medial prefrontal cortex (mPFC). We measured the behavioural effects of housing adult male rats in pairs or alone for 10 days. We also used RNA sequencing to measure the accompanying gene expression alterations in the mPFC of male rats. The isolated animals exhibited reduced sociability and social novelty preference, but increased social interaction and heightened locomotion. There was no change in their aggression, anxiety, or depression-like activity. Transcriptomics analysis revealed differential expression of 46 genes between the groups, many of which have social functions. The KEGG pathway analysis showed that differentially expressed genes are involved in neuroactive ligand-receptor interaction, particularly in the dopaminergic and peptidergic systems and addiction. Subsequent validation confirmed the decreased level of three altered genes: Regulator of G Protein Signalling 9 (Rgs9), Serotonin Receptor 2c (Htr2c), and Prodynorphin (Pdyn), which are involved in dopaminergic, serotonergic, and peptidergic function, respectively. Antagonizing Htr2c confirmed its role in social novelty discrimination. The findings imply that 10 days of social isolation can significantly affect gene expression affecting monoaminergic and peptidergic systems of the mPFC, which enhances our understanding of the effects of social isolation at the molecular level.