A growing body of experimental and clinical studies supports a strong association between psychological stress and cardiovascular disease. An important endogenous cardioprotective role in heart physiology has been attributed to corticotropin-releasing factor receptor type 2beta (CRFR2beta). Here, we report the isolation of cDNA from mouse (m) heart encoding a novel CRFR2beta splice variant. Translation of this insertion variant (iv)-mCRFR2beta isoform produces a 421-aa protein that includes a unique C-terminal cytoplasmic tail. Our functional analysis and cellular localization studies demonstrated that when coexpressed with wild-type mCRFR2beta, iv-mCRFR2beta significantly inhibited the wild-type mCRFR2beta membrane expression and its functional signaling by ER-Golgi complex retention, suggesting a dose-dependent dominant negative effect. Interestingly, mice exposed to a 4-wk paradigm of chronic variable stress, a model of chronic psychological stress in humans, presented significantly lower levels of mCRFR2beta and higher levels of iv-mCRFR2beta mRNA expression in their hearts, compared to nonstressed control mice. The dominant-negative effect of iv-mCRFR2beta and its up-regulation by psychological stress suggest a new form of regulation of the mCRFR2beta cardioprotective effect and a potential role for this novel isoform in stress-induced heart disease.