Objective Leukocyte telomere length (LTL) is a biomarker of cellular aging affected by chronic stress. The relationship of LTL to the stress hormones, cortisol and catecholamines, is unclear, as are possible differences between healthy controls (HC) and individuals with Major Depressive Disorder (MDD). This small pilot study is the first to examine the relationship between cortisol, catecholamines and LTL specifically in un-medicated MDD in comparison with HC. Methods Participants included 16 un-medicated MDD subjects and 15 HC for assay of LTL, 12-hour overnight urinary free cortisol and catecholamine levels. Results LTL, cortisol and catecholamine levels did not significantly differ between groups. In HC, a hierarchical regression analysis indicated that higher levels of cortisol were correlated with shorter LTL (p = 0.003) above and beyond age and sex. Higher catecholamine levels were nearly-significant with shorter LTL (p = 0.055). Neither hormone was correlated with shorter LTL in MDD (p's > 0.28). To assess a possible cumulative effect of stress hormone activation, a summary score was calculated for each subject based on the number of stress hormone levels above the median for that group (HC or MDD). A significant inverse graded relationship was observed between LTL and the number of activated systems in HC (p = 0.001), but not in MDD (p = 0.96). Conclusion This pilot study provides preliminary evidence that stress hormone levels, especially cortisol, are inversely related to LTL in HC, but not in un-medicated MDD. Clarification of these relationships in larger samples could aid in understanding differential mechanisms underlying stress-related cellular aging in healthy and depressed populations.