The intestinal mucosa is constantly exposed to a variety of microbial species including commensals and pathogens, the latter leaving the host susceptible to infection. Antimicrobial peptides (AMP) are an important part of the first line of defense at mucosal surfaces. Human β-defensins (HBD) are AMP expressed by colonic epithelial cells, which act as broad spectrum antimicrobials. This study explored the direct and indirect effects of green kiwifruit (KF) on human β-defensin 1 and 2 (HBD-1 and 2) production by epithelial cells. In vitro digestion of KF pulp consisted of a simulated gastric and duodenal digestion, followed by colonic microbial fermentation using nine human faecal donors. Fermenta from individual donors was sterile filtered and independently added to epithelial cells prior to analysis of HBD protein production. KF products obtained from the gastric and duodenal digestion had no effect on the production of HBD-1 or 2 by epithelial cells, demonstrating that KF does not contain substances that directly modulate defensin production. However, when the digested KF products were further subjected to in vitro colonic fermentation, the fermentation products significantly up-regulated HBD-1 and 2 production by the same epithelial cells. We propose that this effect was predominantly mediated by the presence of short-chain fatty acids (SCFA) in the fermenta. Exposure of cells to purified SCFA confirmed this and HBD-1 and 2 production was up-regulated with acetate, propionate and butyrate. In conclusion, in vitro colonic fermentation of green kiwifruit digest appears to prime defense mechanisms in gut cells by enhancing the production of antimicrobial defensins.