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Aims/Purpose: Pseudoexfoliation glaucoma (XFG) is an aggressive form of secondary open angle glaucoma, associated with activation of the TGF‐β pathway by TGF‐β1. MicroRNAs are small non‐coding genes that regulate mRNA expression. To investigate the role of TGFβ1 in glaucomatous changes in the trabecular meshwork (TM) and to determine how mRNA expression may be regulated by microRNAs in this condition, we performed a bioinformatics analysis to determine the microRNA‐mRNA interactome. Methods: Primary human trabecular meshwork cells were obtained from normal donors (n = 4) and treated with TGF‐β1 at 5 ng/mL for 24 h. RNA was extracted and miRNA‐Seq and mRNA‐Seq performed. Differential mRNA and miRNA expression was determined using a bioinformatics pipeline consisting of FastQC, STAR, FeatureCounts, edgeR (miRNA) and DESeq2 (mRNA). Putative interactions between differentially expressed mRNAs and miRNAs, in which the direction of expression of the regulated mRNA was required to be opposite to that of the regulating miRNA, were determined using interaction databases miRTarBase, TarBase, TargetScan and miRWalk. To classify mRNA species by biological function and pathway, gene enrichment was performed using Enrichr. Results: The resulting microRNA‐mRNA interactome consisted of 1202 interactions. Some highly connected microRNAs were hsa‐let‐7e‐5p, hsa‐miR‐20a‐5p, hsa‐miR‐122‐5p, and hsa‐miR‐29c‐3p. Most genes in all enriched biological functions and pathways were indicated to be regulated by microRNAs. The sub‐interactomes of several specific XFG‐related enrichment terms such as oxidative stress, unfolded protein response, signal molecules and ECM remodelling were determined. Conclusions: This work presents a genome‐wide microRNA‐mRNA regulatory network for human TM cells treated with TGF‐β1 and may serve to generate unbiased hypotheses about regulatory functions of miRNAs found to be differentially expressed in XFG. [ABSTRACT FROM AUTHOR]