The influence of the aromatase enzyme on the chronic fat-sparing effects of testosterone requires further elucidation. Our purpose was to determine whether chronic anastrozole (AN, an aromatase inhibitor) treatment alters testosterone-mediated lipolytic/lipogenic gene expression in visceral fat. Ten-month-old Fischer 344 rats (n = 6/group) were subjected to sham surgery (SHAM), orchiectomy (ORX), ORX + treatment with testosterone enanthate (TEST, 7.0 mg/wk), or ORX + TEST + AN (0.5 mg/day), with drug treatment beginning 14 days postsurgery. At day 42, ORX animals exhibited nearly undetectable serum testosterone and 29% higher retroperitoneal fat mass than SHAM animals (P < 0.001). TEST produced a ∼380-415% higher serum testosterone than SHAM (P < 0.001) and completely prevented ORX-induced visceral fat gain (P < 0.001). Retroperitoneal fat was 21% and 16% lower in ORX + TEST than SHAM (P < 0.001) and ORX + TEST + AN (P = 0.007) animals, while serum estradiol (E2) was 62% (P = 0.024) and 87% (P = 0.010) higher, respectively. ORX stimulated lipogenic-related gene expression in visceral fat, demonstrated by ∼84–154% higher sterol regulatory element-binding protein-1 (SREBP-1, P = 0.023), fatty acid synthase (P = 0.01), and LPL (P < 0.001) mRNA than SHAM animals, effects that were completely prevented in ORX + TEST animals (P < 0.01 vs. ORX for all). Fatty acid synthase (P = 0.061, trend) and LPL (P = 0.043) mRNA levels were lower in ORX + TEST + AN than ORX animals and not different from SHAM animals but remained higher than in ORX + TEST animals (P < 0.05). In contrast, the ORX-induced elevation in SREBP-1 mRNA was not prevented by TEST + AN, with SREBP-1 expression remaining ∼117–171% higher than in SHAM and ORX + TEST animals (P < 0.01). Across groups, visceral fat mass and lipogenic-related gene expression were negatively associated with serum testosterone, but not E2. Aromatase inhibition constrains testosterone-induced visceral fat loss and the downregulation of key lipogenic genes at the mRNA level, indicating that E2 influences the visceral fat-sparing effects of testosterone.