Due to protection of oncogenic proteins from degradation and enhancement of glycolytic phosphometabolites for synthetic processes, respectively, heat shock protein 90 (HSP90) and pyruvate kinase type M2 (PKM2) are important proteins for tumor growth. The present study was undertaken to investigate the susceptibility of both proteins and their encoding genes to the chemopreventive agent butyrate in human colon cells. Matched tissue of different transformation stages derived from 20 individual colon cancer patients was used for the experiments. The results of quantitative real-time PCR revealed a moderate increase of HSP90β and PKM2 mRNA in colon tumors (P < 0.01) compared to normal tissues without relation to clinical parameters. The expression pattern could be confirmed for PKM2 protein by Western blot but not for HSP90β. During culturing with butyrate, the amount of PKM2 transcripts decreased in all three tissue types with the strongest effects observed in tumors (median fold decrease 45%, P < 0.05). The protein data have not reflected this influence supposing a more gradual degradation rate due to a longer half-life of PKM2. In contrast, the mRNA expression of HSP90β in normal tissue was found 1.38-fold increased by butyrate (P < 0.05), but not the corresponding protein level. HSP90β expression in adenomas and tumors remained generally insensitive. Only in malignant tissue, however, a significant correlation was found between the individual effects observed on gene and protein expression level. In conclusion, the present study identified PKM2 as a potential direct target of butyrate in neoplastic colon tissue, whereas HSP90β is none of it.