Ticks use a variety of chemical cues to locate hosts, the main cue being carbon dioxide, which is exhaled by hosts. This study sought to experimentally determine whether ticks exhibit preference among human hosts based on host sex, as the chemical components of human male and female breath have been shown to differ. We focused on the lone star tick, Amblyomma americanum, due to its importance as a disease vector in the United States and its active host-seeking behavior. To test the hypothesis that ticks exhibit preference based upon host sex, we conducted a binary choice behavioral bioassay. Male and female human volunteers (n = 20 pairs) breathed into opposite sides of a secured polycarbonate tube containing 10 adult A. americanum and the proportion of ticks that exhibited a host preference was recorded. We found that under controlled conditions, human females attract a significantly larger proportion of ticks than males. Possible mechanisms to explain these results include that (1) female breath contains components that ticks find attractive, and/or (2) male breath contains a repellent chemical component.