Research indicates that low testosterone is associated with CAD in men. Evidence suggests that men with hypotestosteronemia and concomitant CAD may benefit from physiological testosterone replacement therapy (PTRT). The mechanism by which testosterone produces these cardio-protective effects and the role of the androgen receptor remains largely unknown. The aim of this study was to determine whether testosterone modulates atheroma formation via its classical signalling pathway, via conversion to 17β-estradiol or via an alternative-signalling pathway. Group 1: 8-week-old Tfm (exhibiting a dysfunctional androgen receptor and testosterone deficiency) and control mice were castrated or sham-operated. Group 2: 9-week-old Tfm and controls were administered either placebo, PTRT, PTRT in conjunction with ERα-antagonist or Anastrazole. At 10-weeks both groups were administered a cholesterol-enriched-diet. Mice were sacrificed at 28-weeks. Sections through the aortic sinus were stained using oil-red-O, and lipid-stained areas quantified via digital analysis, and expressed as percentage of medial area. Total cholesterol, HDLC, testosterone and 17β-estradiol were quantified via ELISA. Low endogenous testosterone was associated with fatty-streak formation following feeding on cholesterol-enriched-diet. PTRT prevented aortic fatty streak formation in the Tfm mouse, and increased levels of HDLC. Fatty-streak formation was less marked in PTRT-treated mice, in conjunction with ERα-antagonist or Anastrazole, although this was still significantly lower than that of placebo-treated Tfm mice. Improvement in HDLC was completely attenuated by co-treatment with these agents. PTRT in the Tfm mouse is associated with a reduction in aortic fatty-streak formation. The majority of this action is due to a direct non-genomic action of testosterone, with a component of the response being medicated via conversion to 17β-estradiol and subsequent activation of ERα. The beneficial effect of PTRT upon HDLC appears to be solely mediated by conversion of testosterone into 17β-estradiol, via modulation of genomic ERα-dependent pathways.