BACKGROUND: We previously showed in a rat model of heart failure with preserved ejection fraction (HFpEF) that transcutaneous vagus nerve stimulation (tVNS) reduced cardiac fibrosis and inflammation. However, macrophage-mediated mechanisms through which tVNS rescues cardiac function remain poorly understood. METHODS: We induced HFpEF in 8-week-old mice by a combination of a high-fat diet and l-NG-nitro arginine methyl ester for 5 weeks, followed by 4 weeks of tVNS or sham stimulation. At this time, we analyzed cardiac function by echocardiography and immune cell numbers by single-cell RNA sequencing and flow cytometry. RESULTS: Our data demonstrate that HFpEF mice exhibited diastolic dysfunction, left ventricular hypertrophy, and fibrosis, consistent with HFpEF, and that tVNS significantly improved HFpEF severity. Analysis of merged single-cell RNA sequencing data from control, HFpEF+sham, and HFpEF+tVNS mice showed that HFpEF was associated with the accumulation of Spp1-expressing CCR2 (C-C chemokine receptor type 2)(+) cardiac resident macrophages (CRM). Furthermore, treatment with tVNS reduced the number of CCR2(+) CRM and the expression of Spp1 while also inducing the expression of Igf1 in TLF(+) (Timd4(+) [T-cell immunoglobulin and mucin domain containing 4(+)]/Lyve1(+) [Lymphatic vessel endothelial hyaluronan receptor 1(+)]/Folr2(+) [Folate receptor 2(+)]) and MHC2(+)(Major histocompatibility complex 2(+)) CRM. Global deletion of Spp1 or blockade of CCR2(+) CRM recruitment improved HFpEF, whereas TLF(+)/MHC2(+) specific deletion of Igf1 reversed the protective effect of tVNS on HFpEF. The benefits of tVNS were also abolished in the setting of disrupted acetylcholine/α7nAChR (α7 nicotinic acetylcholine receptor) signaling, either via pharmacological inhibition of α7nAChR or choline acetyltransferase deletion in CD4(+) (cluster of differentiation) T cells. CONCLUSIONS: Collectively, our data indicate that tVNS improves HFpEF by reducing Spp1 expressing CCR2(+) CRM and inducing expression of proreparative Igf1 in TLF(+)/MHC2(+) CRM. These effects are mediated through cholinergic signaling, highlighting a neuroimmune pathway in HFpEF.