This study examined the influence of psychological inflexibility (experiential avoidance) and anxiety sensitivity on psychological and personality variables associated with fibromyalgia syndrome (FMS), as well as on pain perception and heart rate variability (HRV), an indicator of emotional regulation. Thirty-eight FMS patients and thirty-seven healthy controls completed assessments of experiential avoidance, anxiety sensitivity (physical, cognitive, and social), alexithymia, and Eysenck’s personality traits (neuroticism, extraversion, psychoticism) in a single session. Therefore, pain perception was evaluated using pressure algometry and the McGill Pain Questionnaire (MPQ), and HRV was recorded via electrocardiogram. FMS patients showed higher experiential avoidance and greater anxiety sensitivity. They also exhibited higher neuroticism and more difficulties identifying and expressing emotions. Additionally, they demonstrated lower pain thresholds and tolerance compared to controls. HRV values were also lower in the FMS group, indicating a reduced physiological capacity for emotional regulation. In FMS patients, greater experiential avoidance was associated with higher neuroticism and alexithymia, as well as lower pain tolerance. Regarding anxiety sensitivity—particularly its cognitive dimension—higher levels were linked to greater alexithymia, lower HRV, and reduced pain tolerance. In healthy controls, although experiential avoidance showed some associations with alexithymia and neuroticism, it was not related to pain perception or HRV. Psychological and emotional variables, particularly neuroticism, mediated the relationship between experiential avoidance and anxiety sensitivity (predictors) and pain tolerance (outcome). Overall, the findings indicate that FMS patients display a more vulnerable emotional and physiological profile, characterized by heightened pain sensitivity and reduced emotional regulation capacity. Psychological inflexibility and anxiety sensitivity may play a significant role in the maintenance and intensity of pain in FMS, although their impact on pain appears to be largely driven by their association with neuroticism as the key mediating factor.