Introduction: Type 2 diabetes mellitus (T2DM)-associated cardiac fibrosis contributes to heart failure, especially in women. We have data showing that diabetic mice with cardiomyopathy, including cardiac fibrosis, exhibit low levels of the neuropeptide substance P (SP); and SP replacement reverses cardiac fibrosis, independent of body weight and blood glucose. To understand the potential translational significance of these findings, we sought to determine in a T2DM monkey model if replacement SP could reverse cardiac magnetic resonance (CMR) imaging and circulating markers of fibrosis, while also assessing safety of administration. Methods: Female T2DM African Green monkeys were randomized to receive SP (n=4, 0.5 mg/Kg/day S.Q. injection) or vehicle (n=3) for 10 weeks. We obtained CMR imaging and blood samples to assess left ventricular (LV) function and fibrosis by T1 map-derived extracellular volume (ECV) and circulating procollagen type I C-terminal propeptide (PICP). Hematological parameters for toxicities were also assessed. We performed a blinded paired analysis of all parameters. Results: Monkeys receiving replacement SP exhibited a decrease in ECV (Pre: 31.6 ± 5.53% vs. Post: 25.2 ± 3.3%, p=0.03), concomitant with decreased PICP levels (Pre: 5148.58 ± 1143.5 ng/mL vs. Post: 3851.1 ± 1377.5 ng/mL, p=0.01). Animals receiving vehicle showed no changes in ECV (p=0.32) or PICP (p=0.16). No changes in LV ejection fraction were observed (p=0.89). Complete blood counts, metabolic panel (ΔA1c, SP: -0.45 ± 0.59 % vs vehicle: -0.53 ± 0.61 %, p=0.43), lipids, liver and pancreatic enzymes, and inflammation markers were unchanged (p>0.05 for all). Conclusion: Replacement SP reversed cardiac fibrosis in a large preclinical model of T2DM, independent of glycemic control. No organ-related toxicity was associated with replacement SP. These results strongly support a potential application for replacement SP as an antifibrotic therapy for diabetic cardiac fibrosis.