Background Hepatic encephalopathy (HE) is a serious complication of liver cirrhosis. Recently, a microsatellite in the promoter region of the phosphate‐activated glutaminase (GLS ) gene was associated with the risk of HE. The aim of the present study was to investigate, using the critical flicker frequency (CFF) test, whether the described GLS variant increases the risk of developing HE in patients with cirrhosis. Methods We recruited 158 patients (66% men; mean age 59 years, range 23–86) with liver cirrhosis. Mean model for end‐stage liver disease score was 13.8 (range 5–35); 48% of patients presented with Child–Pugh score B or C. The presence and severity of HE were determined by the CFF test, with frequencies ≤39 Hz denoting cases. GLS variants were genotyped by sequencing the microsatellite in the promoter region and were classified as short, long or short–long forms (depending on the length of the macrosatellite alleles). Results In total, 53% of patients had abnormal CFF results (i.e. ≤39 Hz; range for entire cohort 26–57). The GLS microsatellite distribution amongst patients was short form (20%), long form (32%) and short–long form (48%) and was consistent with Hardy–Weinberg equilibrium. CFF values differed significantly between groups (P = 0.043). Carriers of the GLS long microsatellite had a significantly higher risk of HE according to multivariate analyses (odds ratio 3.23, 95% confidence interval 1.46–7.13, P = 0.004). Conclusion CFF results were significantly lower amongst carriers of the GLS long microsatellite. Our findings support the role of the GLS long microsatellite in the development of HE; this could be important for identifying susceptible patients and for the prevention of this condition.