Lower heart rate variability (HRV) and higher inflammation are indices of dysregulated stress physiology associated with current depression (e.g., diagnosis, depressive symptoms) and potentially related to depression development. Given the increasing biological vulnerabilities associated with age, physiological stress sensitivity may be particularly relevant in the development of depression among older adults. While existing meta-analyses report a small-to-medium effect size between HRV and depression severity in unmedicated depressed adults, conflicting findings contest that this association is driven by the cardiovascular effects of antidepressant use. Additionally, findings from at least one longitudinal study suggest that lower HRV may be more strongly linked to depression in males compared to females. Few studies have explored sex differences in HRV and depression, and no longitudinal studies exist examining the relationships among inflammation, HRV, and depression. In a national sample of middle-aged and older adults (MIDUS), this study examined the extent to which sex as well as inflammation moderated the relationship between HRV and concurrent depression (n = 158) as well as future depression development in a larger longitudinal sample (n = 591). Controlling for inflammation, higher high frequency (HF)-HRV significantly predicted less severe depressive symptoms in both medicated (β = -.222, p = .036) and unmedicated cross-sectional samples (β = -.259, p = .044). There was no significant moderation by sex though post-hoc analyses revealed this relationship was driven by females (β = -.329, p = .029). There was no evidence of significant moderation by inflammation. While higher HF-HRV did not significantly predict new incidence of depression in the longitudinal analyses, post-hoc analyses suggest that higher HF-HRV may distinguish between subclinical and clinical major depression among those endorsing past-year depression (OR = .567, p = .010). These findings suggest that dysregulated stress physiology is associated with greater depression severity independent of antidepressant use, particularly among females. Autonomic dysregulation may further predict risk of developing a major depressive episode among middle-aged and older adults endorsing depression.