Hoodia gordonii is currently commercially available as a popular weight-loss supplement. The perceived active ingredient isolated from this plant is a steroidal glycoside named P57. This study investigated the in vitro transport of P57 across excised porcine intestinal and buccal tissue in a Sweetana-Grass diffusion apparatus. In addition, the transport of pure P57 was compared to that obtained from a crude plant extract. Bi-directional transport experiments were conducted in two different media namely Krebs-Ringer bicarbonate buffer and simulated intestinal fluid across intestinal tissue, while apical-to-basolateral transport was conducted in buffer and artificial saliva across buccal tissue. The apparent permeability coefficient (Papp) and flux (J) values were calculated and statistically analysed. The transport of pure P57 across intestinal tissue was significantly higher in the secretory than in the absorptive direction indicating efflux by active membrane transporters. Higher intestinal transport was obtained for P57 in both directions when applied in the form of a crude extract, possibly due to inhibition of efflux. For the buccal tissue, no transport was obtained for the pure P57, while relatively high transport was obtained when applied in the form of a crude extract. Furthermore, the intestinal transport of P57 was significantly decreased when the crude extract was prepared in simulated intestinal fluid compared to the preparation in buffer. Buccal transport was higher in artificial saliva than in buffer. It is therefore evident that the transport of P57 across mucosal tissues is significantly affected on exposure to conditions simulating the in vivo situation.