The transforming growth factor β (TGFβ) family plays a role in fibrosis and has been involved in inflammatory bowel disease (IBD), a chronic gastrointestinal (GI) tract disease that can affect both sexes. Importantly, this is increasingly prevalent in industrialized countries due to changes in lifestyle and diets. The family comprises three isoforms: TGFβ1, TGFβ2 and TGFβ3. While TGFβ1 has an established role in fibrosis, the pathophysiological relevance of the two other isoforms is unclear. Here we evaluated the possible functional and structural alterations of the GI tract and sex influence in an experimental model with partial deficiency of TGF-β3, due to lethality of the homozygous null mouse. Wild-type (WT) and heterozygous for TGF-β3 (HZ) mice of both sexes were exposed to control or high fat diet (HFD), as a possible model of IBD. After oral administration of a radiopaque marker, faeces were collected for 4 h, weighed and exposed to X-rays for GI transit evaluation. Body weight, size/length and histology of the GI organs were also evaluated. No typical signs of IBD were detected. WT females presented lower body weight, delayed GI transit and an increased relative size of the GI organs when compared to males. The HZ genotype modified the latency of expulsion of marked faeces, in a sex and diet dependent manner, without producing macroscopic structural alterations in the GI tract. Moreover, submucosa thickness was decreased in HZ male mice under control diet. HFD increased body weight, accelerated GI transit and decreased GI organs size, especially in females. Importantly, HFD partly counteracted the effects of TGF-β3 heterozygosity on the latency of marked faeces expulsion. To conclude, sex, diet and TGF-β3 genotype alter the GI tract motility and structure, with a possible impact on the IBD development associated with obesity, yet to be determined.