The chemotherapeutic drug substance doxorubicin has been reported to be a substrate of P-gp, which induces a barrier for oral administration and leads to a bioavailability of 3% in male Sprague Dawley rats. Literature studies have reported increased transport of P-pg substrates, like digoxin, when co-administered with P-gp inhibitors (non-ionic surfactants) in vitro and in vivo. The aim of the present study was thus to investigate if different non-ionic surfactants would have a similar effect on the in vitro and in vivo absorption of doxorubicin. This was investigated in vitro in Caco-2 cells and by oral co-administration of doxorubicin together with tween 20 to male Sprague Dawley rats. 200 μM (0.025%) tween 20 increased the intestinal absorptive permeability of doxorubicin in vitro by 48 ± 4% from 8.8 × 10−6 cm/s to 13.0 × 10−6 cm/s. Further, the efflux ratio was reduced from 2.2 ± 0.06 to 1.2 ± 0.03 (n = 3–7). In vivo, co-administration of doxorubicin and 0–25% tween 20 did not yield detectable doxorubicin plasma concentrations, probably due to extensive intestinal metabolism. In conclusion, the present study demonstrated that non-ionic surfactants increased the transport of doxorubicin in vitro, most likely by inhibition of the efflux activity. However, this effect was not transferable to the in vivo situation.