Gold nanoparticles (GNPs) have gained considerable attention for application in science and industry. However, the untoward effects of such particles on female fertility remain unclear. The objectives of this study were to (1) examine the effects of 10-nm GNPs on progesterone and estradiol-17β accumulation by rat ovaries ex vivo and (2) to identify the locus/loci whereby GNPs modulate steroidogenesis via multiple-reference gene quantitative real-time RT-PCR. Regression analyses indicated a positive relationship between both Star (p < 0.05, r(2) = 0.278) and Cyp11a1 (p < 0.001, r(2) = 0.366) expression and P4 accumulation upon exposure to 1.43 × 10(6) GNPs/mL. Additional analyses showed that E2 accumulation was positively associated with Hsd3b1 (p < 0.05, r(2) = 0.181) and Cyp17a1 (p < 0.01, r(2) = 0.301) expression upon exposure to 1.43 × 1(3) and 1.43 × 10(9) GNPs/mL, respectively. These results suggest a subtle treatment-dependent impact of low-dose GNPs on the relationship between progesterone or estradiol-17β and specific steroidogenic target genes, independent of oxidative stress or inhibin.