The RNA-binding protein HuR regulates the stability and translation of numerous mRNAs encoding stress-response and proliferative proteins. Although its post-transcriptional influence has been linked primarily to its cytoplasmic translocation, here we report that moderate heat shock (HS) potently reduces HuR levels, thereby altering the expression of HuR target mRNAs. HS did not change HuR mRNA levels or de novo translation, but instead reduced HuR protein stability. Supporting the involvement of the ubiquitin-proteasome system in this process were results showing that (1) HuR was ubiquitinated in vitro and in intact cells, (2) proteasome inhibition increased HuR abundance after HS, and (3) the HuR kinase checkpoint kinase 2 protected against the loss of HuR by HS. Within a central, HS-labile approximately 110-amino-acid region, K182 was found to be essential for HuR ubiquitination and proteolysis as mutant HuR(K182R) was left virtually unubiquitinated and was refractory to HS-triggered degradation. Our findings reveal that HS transiently lowers HuR by proteolysis linked to K182 ubiquitination and that HuR reduction enhances cell survival following HS.