Hyperglycaemia is a characteristic of type 2 diabetes. In hepatocytes, impaired insulin sensitivity leads to increased gluconeogenesis and decreased glycogenesis. MicroRNA (miR)‑338‑3p is associated with tumour necrosis factor (TNF)‑α‑induced suppression of hepatic glycogenesis via regulation of protein phosphatase 4 regulatory subunit 1 (PP4R1). However, the effect of miR‑338‑3p on gluconeogenesis in hepatocytes remains unknown. In a previous study, it was demonstrated that miR‑338‑3p is downregulated in the livers of mice and in mouse HEPA1‑6 hepatocytes following treatment with TNF‑α. In the present study, the effect of miR‑338‑3p on TNF‑α‑induced gluconeogenesis in hepatocytes was investigated. The levels of phosphorylated‑FOXO1/FOXO1, phosphoenolpyruvate carboxykinase (PEPCK), peroxisome proliferator‑activated receptor γ coactivator (PGC‑1α) and glucose‑6‑phosphatase (G6Pase) were measured by western blotting. The mRNA levels of PEPCK, PGC‑1α and G6Pase were determined by quantitative polymerase chain reaction. Pyruvate tolerance testing was used to determine the gluconeogenesis of mouse livers. The results demonstrated that treatment with TNF‑α resulted in increased levels of gluconeogenesis in the livers of mice and decreased miR‑338‑3p expression levels in HEPA1‑6 cells. Overexpression of miR‑338‑3p reversed TNF‑α‑induced glucose production via enhancement of phosphorylated forkhead box O1 levels and downregulation of the expression levels of genes associated with gluconeogenesis, including peroxisome proliferator‑activated receptor γ coactivator‑1α, phosphoenolpyruvate carboxykinase and glucose‑6‑phosphatase. However, inhibition of miR‑338‑3p expression was revealed to enhance gluconeogenesis in the livers of mice and in HEPA1‑6 cells. Furthermore, downregulation of PP4R1 was revealed to attenuate the effect on glucose production following treatment with miR‑338‑3p inhibitors. In conclusion, the results of the present study revealed that miR‑338‑3p may be involved in TNF‑α‑mediated gluconeogenesis via targeting of PP4R1 in hepatocytes.