Histone deacetylase inhibitors (HDACIs) are emerging as a novel class of antitumor drugs. But the effect of HDACIs in tumors treatment has been disappointing, which mainly due to the acquisition of resistance to HDACIs. However, the underlying mechanisms have not been clearly understood. In this study, it was found that HDACIs SAHA and TSA increased P-gp expression in CRC cells, which has been well known to contribute to drug resistant. The mechanisms underlying these effects were investigated. We showed that HDACIs enhanced transcriptional activity of P-gp protein encoding gene ABCB1. HDACIs treatment also increased the protein and mRNA expression of STAT3, but not PXR, CAR, Foxo3a or beta-catenin, which are known to be involved in ABCB transcription regulation. Interestingly, knockdown of STAT3 significantly attenuated HDACIs-induced P-gp up-regulation in colorectal cancer cells, suggesting that STAT3 plays a crucial role in HDACIs-up-regulated P-gp. Furthermore, this study revealed for the first time that HDACIs enhanced the stability of ABCB1 at post-transcriptional level. Taken together, these results proved that HDACIs induced P-gp expression by two distinct ways, transcriptional activation and mRNA stabilization. Our results suggested that more attention should be paid to the cancer treatment using HDACIs since they will induce multidrug resistance in cancer cells.