We investigated methods for reducing highway-caused mortality of Mojave Desert Tortoises (Gopherus agassizii) using barriers at the Desert Tortoise Conservation Center, located near Las Vegas, Nevada. Experiments tested effectiveness of various barriers of different heights and made of different construction materials, sex-dependent behaviors including rate of escapes, appropriate height of a barrier, and adverse effects on other sympatric vertebrate species including potential entrapment. Informed by analyzing previous investigations, we tested captive adult desert tortoises (midline carapace length ≥ 180 mm) within enclosures made from 8 different common construction materials, quantified behavioral responses of individuals over several (4–14) days, and identified sex-dependent responses to these barriers. Both sexes pushed against see-through barriers more than solid barriers (58% males, 67% females) and males reversed direction of pacing more often than females (56%). Pen escapes related to pacing reversals and were generally by males. Direction reversals and escapes occurred mostly with wire mesh designs and low (20-cm) solid barriers. Frequency of body contact was not linearly correlated with larger mesh sizes. Solid barriers appear to offer the best deterrent but are more expensive. Animals that escaped in trials lasting several days were larger than average size and often escaped more than once. Tortoises escaped at weak points in barriers, such as gaps with the ground, pen corners, breaks in material, or by climbing over the barrier. In testing minimum effective barrier height for 3 types of barrier materials, none climbed over at 46 cm, 5 climbed over at 30 cm, and 19 others climbed over at lower heights. We observed native species interactions with our barrier enclosures (n = 70) and compared morphometric measurements of common desert reptiles vs. barrier dimensions. These data suggested that different barrier types preferentially affect specific sizes of animals although we observed no mortality. While solid barriers stop all reptiles, wire meshes entrap reptile species smaller than mesh size (such as Callisaurus draconoides and Uta stansburiana) and stop larger species (such as Dipsosaurus dorsalis and Coluber flagellum). Morphometric measures of juvenile tortoises indicated that they could pass through the 2 largest wire mesh sizes we tested.