Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. It is a spectrum of disease ranging from simple intracellular lipid accumulation and eventually progressing to cirrhosis and hepatocellular carcinoma (HCC). 5β-reductase (AKR1D1) is highly expressed in human liver and catalyzes a fundamental step in bile acid (BA) synthesis. BAs are established as potent regulators of metabolic phenotype and we have hypothesised that AKR1D1 plays a crucial regulatory role in NAFLD and HCC. Human liver biopsies were obtained from 34 obese patients. Genetic manipulation of AKR1D1 was performed in human HepG2 and Huh7 hepatocellular carcinoma cell lines. mRNA and protein expression changes were determined using qPCR, RNA-sequencing and western blotting. Effects on cell proliferation and cell cycle were determined using CyQuant, flow cytometry, transcriptome analysis, and enzyme immunoassays. In human liver biopsies, AKR1D1 expression decreased with advancing steatosis, inflammation and fibrosis and was significantly reduced in patients with type 2 diabetes. In HepG2 cells, RNA sequencing analysis, following transient AKR1D1 knockdown, identified discrete dysregulated pathways impacting lipid metabolism, DNA replication, cell cycle and p53 signalling. In both cell lines, AKR1D1 knockdown decreased BA synthesis with a consequent downstream decrease in cyclin-dependent kinase (CDK1, CDK2, CDK4, CDK6) and proliferation markers expression (CDC6, PCNA, PLK1). In contrast, expression of cyclin-dependent kinase inhibitors (CDKIs: p15, p16, p21) increased. Endorsing these data, HepG2 cells with stable knockdown of AKR1D1 proliferated more slowly with evidence of cell cycle arrest on flow cytometry. Pharmacological manipulation of BA receptor activation prevented the induction of CDKIs gene expression, suggesting that the observed phenotype is driven, at least in part, through BA and/or oxysterol availability. In conclusion, AKR1D1 is down-regulated with advancing NAFLD and inhibits hepatocellular carcinoma cell proliferation in vitro. Taken together, these data suggest an as yet unexplored role of AKR1D1 in the progression of NAFLD to HCC.