Purpose Mesenchymal stromal cells (MSC) are an attractive tool for treatment of diabetic cardiomyopathy. Syndecan‐2/CD362 has been identified as a functional marker for MSC isolation. Imaging mass spectrometry (IMS) allows for the characterization of therapeutic responses in the left ventricle. This study aims to investigate whether IMS can assess the therapeutic effect of CD362+‐selected MSC on early onset experimental diabetic cardiomyopathy. Experimental Design 1 × 106 wild type (WT), CD362−, or CD362+ MSC are intravenously injected into db/db mice. Four weeks later, mice are hemodynamically characterized and subsequently sacrificed for IMS combined with bottom‐up mass spectrometry, and isoform and phosphorylation analyses of cardiac titin. Results Overall alterations of the cardiac proteome signatures, especially titin, are observed in db/db compared to control mice. Interestingly, only CD362+ MSC can overcome the reduced titin intensity distribution and shifts the isoform ratio toward the more compliant N2BA form. In contrast, WT and CD362− MSCs improve all‐titin phosphorylation and protein kinase G activity, which is reflected in an improvement in diastolic performance. Conclusions and Clinical Relevance IMS enables the characterization of differences in titin intensity distribution following MSC application. However, further analysis of titin phosphorylation is needed to allow for the assessment of the therapeutic efficacy of MSC.