The role of immune cells in the central nervous system and their interaction with peripheral monocytes are extensively investigated in the recent decade. Both animal models of parkinsonism and post-mortem studies demonstrated that peripheral immune cell recruitment occurs due to neuroinflammation seen in the Parkinson's disease pathology. Understanding these interaction mechanisms and possible modulators that have played a role in these processes is important for treatment options. As an immune anchor of the central nervous system, microglia are suggested to be one of the possible modulators of this interaction. IL-16 was recently described as a novel regulator for T cell recruitment and probably had a role in sporadic Parkinson's disease seen in the Chinese population. With the knowledge of epigenetic alterations in Parkinson's disease is seen in the immune cells, in the present study, we investigated possible changes in IL-16 levels with or without lenalidomide treatment in the rotenone-induced N9 microglial cells by enzyme-like immunosorbent assay (ELISA). Additionally, we also evaluated the methylation status of the IL-16 gene promoter after drug treatments by methylation specific-polymerase chain reaction (ms-PCR) analysis. Our results indicate that rotenone causes an increase in the IL-16 release, and lenalidomide attenuates that release in microglial cells. Additionally, rotenone changes the methylated status of IL-16 to unmethylated status, which explains increased IL-16 levels. However, lenalidomide treatment inhibited an increase in rotenone-induced IL-16 levels. Therefore, the present study suggests that lenalidomide improves the methylation status of IL-16 and prevented the increase in the IL-16 levels due to the rotenone insult.