Efficacy of many novel therapeutic agents are impaired by hindered interstitial diffusion in tumor. In the context of overcoming this drug delivery barrier, a hypothesis was postulated that freeze/thaw (F/T) may induce favorable changes of tumor tissue microstructure to facilitate the interstitial diffusion. This hypothesis may also be relevant to develop a mechanistically derived chemotherapeutic strategy for cryo-treated tumors. In the present study, this hypothesis was tested by characterizing the effects of F/T on the interstitial diffusion using an in vitro engineered tumor model (ET). The diffusion coefficients of FITC-labeled dextran was measured within the frozen/thawed and unfrozen ETs. The results showed that the diffusion coefficients increased after F/T but the extent of increase was dependent on the size of dextran. This implies that the combination of cryosurgery and chemotherapy should be designed considering the biophysical changes of tissues after freeze/thaw and the diffusion characteristics of drug molecules.