Abstract Body: Heart disease remains a leading cause of death globally, often accompanied by conditions such as coronary artery disease and hypertension. The vasodilator nitric oxide (NO) is crucial for blood pressure control and is regulated through metabolism to nitrate by hemoglobin. Another globin termed cytoglobin (Cygb), expressed in medial vascular smooth muscle cells, has also been proposed to play a significant role in regulating blood pressure through NO metabolism. However, the role of Cygb in regulating hypertension-induced organ damage, such as cardiac hypertrophy and fibrosis, is unknown. Thus, this study aims to explore the role of Cygb in angiotensin II (Ang II)-induced cardiac hypertrophy in the mouse. We hypothesized that Cygb plays a pro-hypertensive role and promotes cardiac hypertrophy. Methods: We used global Cygb knockout (KO) and wildtype littermate mice treated with Ang II (1.5 mg/kg/day) for two weeks via subcutaneous osmotic minipumps to induce cardiac hypertrophy. Both females and males were used in these studies. Cardiac function was assessed through echocardiography, and hearts were analyzed for changes in hypertrophy, fibrosis, and gene expression. Results: Contrary to our initial hypothesis, male Cygb KO mice showed increased cardiac hypertrophy and reduced ejection fraction following Ang II infusion rather than the expected protective effect. Importantly, loss of Cygb did not affect systemic blood pressure in mice, either at baseline or after Ang II treatment, and irrespective of sex. There was also no change in acetylcholine-mediated vasorelaxation in isolated vessels. Single-cell transcriptomic analysis indicated that Cygb was predominantly expressed in cardiac fibroblasts, with immunofluorescent staining localizing the protein mainly to interstitial and perivascular fibroblasts within the heart. Differential gene expression analysis revealed that Cygb-deficient hearts exhibited an increased cellular response to interferon-gamma and a diminished response to TGF-beta signaling. Conclusions: Our findings indicate that Cygb plays a protective role in the mouse heart during Ang II-induced cardiac stress. The increased hypertrophy in Cygb KO mice suggests that fibroblast Cygb may be crucial for preserving cardiac function, possibly through modulation of inflammatory and pro-fibrotic pathways.