Background: The potential molecular mechanisms underlying acute adaptation and chronic remodeling of cardiac structure and function in response to high altitude are not yet fully understood. This study aims to investigate changes in the crotonylproteome in the hearts of mice exposed to high altitude at different time points. Methods: The hearts were obtained from mice living at the lowland (500 meters above the sea level, Control), and 3- (D3), 10- (D10) and 30-days (D30) after arriving at plateau (3600 meters above the sea level). Then, a high-resolution mass spectrometry-based, quantitative crotonyllysine proteomics approach was performed to identify the changes in crotonylproteome. Results: We totally identified approximately 13,731 crotonyllysine sites, among which 11,140 were quantified. Furthermore, 1247 differentially expressed (DE) crotonyllysine sites on 764 proteins in 46 pathways were defined by using a stepwise pipeline. Then, 90 crotonylosites from 88 crotonyloproteins with increased crotonylosite abundance and 185 crotonylosites from 141 crotonyloproteins with decreased crotonylosite abundance in the hearts of D3 group were identified, 353 hypercrotonylosites from 274 hypercrotonyloproteins and 118 hypocrotonylosites from 95 hypocrotonyloproteins in the hearts of D10 group were identified, and 958 hypercrotonylosites from 638 hypercrotonyloproteins and 77 hypocrotonylosites from 63 hypocrotonyloproteins were identified in the hearts of D30 group when compared to that in control groups, respectively (>1.3-fold change, P < 0.05), while which occurs largely independently of protein abundance in the same proteins, suggesting a gradually increased crotonylation abundance with the time of exposure to high altitude. Interestingly, the functional enrichment analysis found that most of these significantly changed crotonylated proteins were enriched in the regulation of metabolism and cardiac muscle functions, indicating that these enriched biological processes may be involved in the acute adaptation and chronic remodeling of cardiac function and structure changes in response to high altitude. Conclusion: Our study uncovers some crotonylation-affected processes and pathways in the hearts response to high-altitude exposure, giving novel insights into molecular processes of cardiac function and structure changes at high altitude.