The activity of natural killer (NK) cells is regulated by activating and inhibiting receptors, whereby the C-type lectin natural killer group 2D (NKG2D) receptor serves as the major activating receptor on NK cells which recognizes major histocompatibility class I chain-related proteins A and B (MICA/B). The MICA/B expression has been described to be regulated by the transcription factor heat shock factor 1 (HSF1). Inhibition of heat shock protein 90 (Hsp90) is known to induce the heat shock response via activation of HSF1 which is associated with tumor development, metastasis and therapy resistance and also with an increased susceptibility to NK cell-mediated lysis. Therefore, we compared the effects of Hsp90 inhibitor NVP-AUY922, HSF1 inhibitor NZ28 and HSF1 knockdown on the sensitivity of lung (H1339) and breast (MDA-MB-231, T47D) cancer cells to NK cell-mediated cytotoxicity and the expression of the NKG2D ligands MICA/B. Although NVP-AUY922 activates HSF1, neither the MICA/B surface density on tumor cells nor their susceptibility to NK cell-mediated lysis was affected. A single knockdown of HSF1 by shRNA decreased the surface expression of MICB but not that of MICA, and thereby, the NK cell-mediated lysis was only partially blocked. In contrast, NZ28 completely blocked the MICA/B membrane expression on tumor cells and thereby strongly inhibited the NK cell-mediated cytotoxicity. This effect might be explained by a simultaneous inhibition of the transcription factors HSF1, Sp1 and NF-κB by NZ28. These findings suggest that new anticancer therapeutics should be investigated with respect to their effects on the innate immune system.