Cordycepin is one of the substantial components of the parasitic fungus Cordyceps sinensis as well as other Cordyceps species. It exerts various effects such as antimetastatic, antiinflammatory, antioxidant, and neuroprotective activities. Assorted studies revealed in vitro and in vivo anticancer influence of cordycepin and put forward its potential for cancer therapy. However, the role of multidrug resistance-associated mechanisms for the antitumor effect of cordycepin has not been investigated in great detail thus far. Therefore, we searched cordycepin's cytotoxicity with regard to well-known anticancer drug resistance mechanisms, including ABCB1, ABCB5, ABCC1, ABCG2, EGER, and TP53, and identified putative molecular determinants related to the cellular responsiveness of cordycepin. Bioinformatic analyses of NCI microarray data and gene promoter transcription factor binding motif analyses were performed to specify the mechanisms of cordycepin towards cancer cells. COMPARE and hierarchical analyses led to the detection of the genes involved in cordycepin's cytotoxicity and sensitivity and resistance of cell lines towards cordycepin. Tumor-type dependent response and cross-resistance profiles were further unravelled. We found transcription factors potentially involved in the common transcriptional regulation of the genes identified by COMPARE analyses. Cordycepin bypassed resistance mediated by the expression of KR-binding cassete (ABC) transporters (P-gp, ABCB5, ABCC1 and BCRP) and mutant epidermal growth factor receptor (EGFR). The drug sensitivity profiles of several DNA Topo I and II inhibitors were significantly correlated with those of cordycepin's activity. Among eight different tumor types, prostate cancer was the most sensitive, whereas renal carcinoma was the most resistant to cordycepin. NF-kappa B was discovered as a common transcription factor. The potential of cordycepin is set forth as a potential new drug lead by bioinformatic evaluations. Further experimental studies are warranted for better understanding of cordycepin's activity against cancer.