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Objective: Insulin resistance is a critical factor contributing to the pathogenesis of type 2 diabetes and other metabolic diseases. Recent studies have indicated that miR-338-3p plays an important role in cancer. Here, we investigated whether miR-338-3p mediates tumour necrosis factor-α (TNF-α)-induced hepatic insulin resistance. Methods: The activation of the insulin signalling pathway and the level of glycogenesis were examined in the livers of the db/db and high fat diet (HFD)-fed mice and in HEP1-6 cells transfected with miR-338-3p mimic or inhibitor. Computational prediction of microRNA target, luciferase assay and Western blot were used to assess the miR-338-3p target. Chromatin immunoprecipitation (ChIP) assay was used to determine the transcriptional regulator of miR-338-3p. Results: miR-338-3p was down-regulated in the livers of the db/db, HFD-fed and TNF-α-treated C57BL/6J mice, as well as in mouse HEP1-6 hepatocytes treated with TNF-α. Importantly the down-regulation of miR-338-3p induced insulin resistance, as indicated by impaired glucose tolerance and insulin tolerance. Further research showed that the down-regulated miR-338-3p resulted in the impaired AKT/ glycogen synthase kinase 3 beta (GSl·Gβ) signalling pathway and glycogen synthesis. In contrast, hepatic over-expression of miR-338-3p rescued the TNF-α-induced insulin resistance. Moreover, protein phosphatase 4 regulator subunit 1 (PP4R1) was identified as a direct target of miR-338-3p that mediated hepatic insulin signalling by regulating protein phosphatase 4 (PP4). Finally we identified hepatic nuclear factor 4 alpha (HNF-4α) as the transcriptional regulator of miRNA-338-3p. Conclusions: Our studies provide novel insight into the critical role and molecular mechanism by which miR-338-3p is involved in TNF-α-induced hepatic insulin resistance. miR-338-3p might mediate TNF-α-induced hepatic insulin resistance by targeting PP4R1 to regulate PP4 expression.