The retinal pigment epithelium (RPE) is central to retinal health and immune regulation. In diseases, such as proliferative vitreoretinopathy (PVR), dysregulated RPE function, driven by aberrant signaling pathways like mitogen-activated protein kinase (MAPK), contributes to fibrotic membrane formation and retinal detachment. Tacrolimus, an immunosuppressive agent, has shown potential to modulate signaling beyond immune cells, but its effect on MAPK signaling in RPE cells remains unclear. This study aimed to investigate the impact of tacrolimus on MAPK pathway gene expression and microRNA (miRNA)-mediated regulation in human RPE (H-RPE) cells under inflammatory conditions induced by lipopolysaccharide (LPS). H-RPE cells were treated with LPS and tacrolimus, and cell viability was evaluated by 3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Transcriptomic profiling of 300 MAPK-related genes and corresponding miRNAs was performed using Affymetrix microarrays. Key targets were validated via quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Gene interaction networks were analyzed with STRING. LPS significantly suppressed MAPK pathway gene and protein expression, including transforming growth factor beta 1 (TGF-β-1), mitogen-activated protein kinase kinase 7 (MAP2K7), mitogen-activated protein kinase 3 (MAPK3), and dual specificity phosphatase 4 (DUSP4). Tacrolimus reversed these effects in a time-dependent manner, restoring expression levels and modulating regulatory miRNAs (e.g., miR-3196, miR-27a/b-3p, miR-190a-3p, miR-149-3p). STRING analysis revealed a highly connected protein network, with MAPK3, MAPK8, and TRAF6 acting as central nodes. Tacrolimus modulates MAPK signaling in H-RPE cells by reversing LPS-induced suppression and regulating specific miRNAs. These findings suggest a potential therapeutic role for tacrolimus in mitigating inflammatory and fibrotic responses associated with PVR.