The aim of the present study was to investigate sex differences in light color preference and the key role of aromatase in the conversion of testosterone (T) to estradiol (E2) in the regulation of this preference in a stream-dwelling fish, Acrossocheilus fasciatus. Six monochromatic lights were tested. The effects of reduced aromatase activity, via drug inhibition, on plasma sex hormone levels (T and E2) and the preferences between sexes were investigated. The results showed that female and male fish preferred red light (around 637 nm in wavelength) and yellow light (590 nm), respectively. However, they were repelled by blue light (465 nm), purple light (405 nm), and white light with blue spectrum (447 nm), while the preference for green light (518 nm) was not obvious. Feeding the fish an experimental diet containing the aromatase inhibitor, letrozole (LZ), over 10 days, resulted in a significant reduction in the aromatase activity level in the brain and gonadal tissues, as well as in the plasma E2 level, but no change in the plasma T level was observed. Surprisingly, LZ also caused an obvious change in the color preference of lighting; i.e., females preferred blue light and males preferred red light after treatment. However, no significant changes occurred in the levels of T and E2; nor were there any in the preferences, except for the decrease in aromatase activity levels in both the females and the males fed with another experimental feed containing LZ+E2. In conclusion, the sex differences in the color preference of lighting in these fish are most likely related to the sex differences in aromatase activity and estradiol levels, but not significantly related to the testosterone levels.