Objective: β-Adrenergic receptor signaling, a critical mediator of sympathetic nervous system influences on physiology and behavior, has long been proposed as one contributor to subjective stress. However, prior findings are surprisingly mixed about whether β-blockade (e.g., propranolol) blunts subjective stress, with many studies reporting no effects. We reevaluated this question in the context of an acute psychosocial stressor with more comprehensive measures and a larger-than-typical sample. We also examined the effects of β-blockade on psychophysiological indicators of sympathetic and parasympathetic nervous system reactivity, given that β-blockade effects for these measures specifically under acute psychosocial stress are not yet well established. Methods: In a double-blind, randomized, placebo-controlled study, 90 healthy young adults received 40 mg of the β-blocker propranolol or placebo. Participants then completed the Trier Social Stress Test, which involved completing an impromptu speech and difficult arithmetic in front of evaluative judges. Self-reported emotions and appraisals as well as psychophysiology were assessed throughout. Results: Propranolol blunted Trier Social Stress Test preejection period reactivity (b = 9.68, p = .003), a marker of sympathetic nervous system activity, as well as salivary α-amylase reactivity (b = −0.50, p = .006). Critically, propranolol also blunted negative, high arousal emotions in response to the stressor (b = −0.22, p = .026), but cognitive appraisals remained intact (b values < −0.17, p values > .10). Conclusions: These results provide updated experimental evidence that β-adrenergic blockade attenuates negative, high arousal emotions in response to a psychosocial stressor while also blunting sympathetic nervous system reactivity. Together, these findings shed light on the neurophysiological mechanisms by which stressors transform into the subjective experience we call "stress." Trial Registration: ClinicalTrials.gov Identifier: NCT02972554.