Resistance to anoikis (matrix deprivation-induced apoptosis) is a critical component of the metastatic cascade. Molecular mechanisms underlying resistance to anoikis have not been reported in thyroid cancer cells. For an in vitro model of anoikis, we cultured follicular, papillary, and anaplastic thyroid cancer cell lines on poly-HEMA-treated low-adherent plates. We also performed immunohistochemical analysis of human cancer cells that had infiltrated blood and/or lymphatic vessels. Matrix deprivation was associated with establishment of contacts between floating thyroid cancer cells and formation of multi-cellular spheroids. This process was associated with activation of gap junctional transfer. Increased expression of the gap junction molecule Connexin43 was found in papillary and anaplastic cancer cells forming spheroids. All non-adherent cancer cells showed a lower proliferation rate compared with adherent cells but were more resistant to serum deprivation. AKT was constitutively activated in cancer cells forming spheroids. Inhibition of gap junctional transfer through Connexin43 silencing, or by treatment with the gap junction disruptor carbenoxolone, resulted in loss of pAKT and induction of apoptosis in a cell-type-specific manner. In human thyroid tissue, cancer cells that had infiltrated blood vessels showed morphological similarity to cancer cells forming spheroids in vitro. Intra-vascular cancer cells demonstrated prominent AKT activation in papillary and follicular cancers. Increased Connexin43 immunoreactivity was observed only in intra-vascular papillary cancer cells. Our data demonstrate that establishment of inter-cellular communication contributes to thyroid cancer cell resistance to anoikis. These findings suggest that disruption of gap junctional transfer could represent a potential therapeutic strategy for prevention of metastases.