Essential fatty acid (EFA) deficiency in mice induces fat malabsorption. We previously reported indications that the underlying mechanism is located at the level of the intestinal mucosa. We have investigated the effects of EFA deficiency on small intestinal morphology and function. Mice were fed an EFA-deficient or control diet for 8 weeks. A 72 h fat balance, the EFA status, and small intestinal histology were determined. Carbohydrate absorptive and digestive capacities were assessed by stable isotope methodology after administration of U-¹³C-glucose and 1-¹³C-lactose. The mRNA expression and enzyme activity of lactase, and concentrations of the EFA linoleic acid (LA) were measured in small intestinal mucosa. Mice fed the EFA-deficient diet were markedly EFA-deficient with a profound fat malabsorption. EFA deficiency did not affect the histology or proliferative capacity of the small intestine. Blood ¹³C₆-glucose appearance and disappearance were similar in both groups, indicating unaffected monosaccharide absorption. In contrast, blood appearance of ¹³C-glucose, originating from 1-¹³C-lactose, was delayed in EFA-deficient mice. EFA deficiency profoundly reduced lactase activity (-58%, p<0.01) and mRNA expression (-55%, p<0.01) in mid small intestine. Both lactase activity and its mRNA expression strongly correlated with mucosal LA concentrations (r=0.89 and 0.79, resp., p< 0.01). EFA deficiency in mice inhibits the capacity to digest lactose, but does not affect small intestinal histology. These data underscore the observation that EFA deficiency functionally impairs the small intestine, which in part may be mediated by low LA levels in the enterocytes.