Objectives: Patients with heart failure (HF) experience severe pain and may have altered pain sensation; however, the underlying mechanisms of these symptoms are not yet fully understood. Identifying pain sensation and genomic biomarkers of pain in older adults with HF is a critical step toward developing personalized interventions to improve pain management and outcomes. This study aimed to investigate the differences in pain sensation, gut microbiota, self-reported pain, and symptoms in older adults with and without HF. Methods: Twenty older adults with HF and age-matched healthy controls (HCs) were recruited in the Northeastern United States. Quantitative sensory testing and conditioned pain modulation were performed on the nondominant upper arm to detect the mechanical, thermal, and pressure pain thresholds and pain modulations. Stool samples were collected, and the 16S rRNA V4 gene region of stool samples was sequenced and processed using the Mothur 1.42.3 pipeline. Self-reported pain and symptoms were measured by the Brief Pain Inventory and the NIH Patient-reported Outcomes Measurement Information System. The associations between pain sensation, gut microbiota α-diversity indices, and pain and symptoms were explored using the Spearman correlations. Results: The HF and HC subjects' mean ages were 73.50 (SD = 8.33) and 67.10 (SD = 7.64), respectively. The HF subjects reported significantly higher pain intensity and interference, sleep disturbance, fatigue, anxiety, and depression than the HCs. The HF subjects also had a significantly lower level of physical function and participation in social roles and activities. Compared with the HCs, the HF subjects had significantly altered conditioned pain modulation heat effect and gut microbiota compositions and predicted metabolic functions. The Statistical Analysis Of Metagenomic Profiles indicated that the HF subjects had a significantly decreased cardiac muscle contraction pathway compared with the HCs. The correlation analysis showed that the quantitative sensory testing profiles and gut microbiota diversity index were significantly associated with pain and symptoms in older adults with HF. Conclusions: Older adults with HF had more severe self-reported pain and symptoms, altered pain sensation, and different gut microbiota composition and function compared with age-matched HCs. Pain sensation and gut microbiota may contribute to pain and symptoms in older adults with HF and could serve as biomarkers of pain and symptoms of HF. Further research with a larger sample size is warranted to confirm these findings.