Aberrant activation of nuclear factor-κB (NF-κB) has been observed in a wide range of human cancers and is thought to promote tumorigenesis and metastasis. As a central component of NF-κB pathway, p65 protein level is tightly regulated and could be subjected to proteasome degradation. Here we demonstrated that p65 can bind to HSC70 with four consensus recognition motif in its RHD domain and be constitutively transported to the lysosome membrane to bind with lysosome-associated membrane protein type 2A and degraded within the lysosome in two epithelial cell lines, proposing that p65 can be degraded by chaperone-mediated autophagy (CMA). Of great importance, there is a decreased CMA activity together with impaired degradation of p65 in a process of epithelial–mesenchymal transition (EMT). The resulted accumulation of p65 leads to higher NF-κB activity and contributes to the progression and maintenance of the EMT program. Taken together, our results define a novel regulatory mechanism for the important transcription factor p65, and these findings would shed new light on the inhibition of EMT, as well as metastasis of cancer cells.