Diabetic patients suffer from gastrointestinal disorders associated with dysmotility, enteric neuropathy and dysbiosis of gut microbiota, however gender differences are not fully known. Previous studies show that high-fat diet (HFD) causes type two diabetes (T2D) in male mice after 4-8 weeks, but only does so in female mice after 16 weeks. This study sought to determine whether sex influences the development of intestinal dysmotility, enteric neuropathy and dysbiosis in mice fed HFD. We fed 8-week old C57BL6 male and female mice a standard chow diet (SCD) or a 72% Kcal HFD for 8 weeks. We analyzed the associations between sex and intestinal dysmotility, neuropathy and dysbiosis using motility assays, immunohistochemistry and next generation sequencing. HFD ingestion caused obesity, glucose intolerance and insulin resistance in male but not female mice. However, HFD ingestion slowed intestinal propulsive motility in both male and female mice. This was associated with decreased inhibitory neuromuscular transmission, loss of myenteric inhibitory motor neurons, and axonal swelling and loss of cytoskeletal filaments. HFD induced dysbiosis and changed the abundance of specific bacteria, especially Allobaculum, Bifidobacterium and Lactobacillus, which correlated with dysmotility and neuropathy. Female mice had higher immunoreactivity and numbers of myenteric inhibitory motor neurons, matching larger amplitudes of inhibitory junction potentials.