Testosterone is a sex hormone involved in brain maturation via multiple molecular mechanisms. Our previous studies have suggested a possible role of this hormone in regulating radial growth of the axon and, in turn, the overall volume of white matter during male puberty. We have proposed that testosterone-induced remodeling of axonal cytoskeleton may be associated with variations in axonal transport. Here we examined the effect of the non-aromatizable androgen mibolerone on the bidirectional transport of wheat-germ agglutinin vesicles in the axons of cultured sympathetic neurons. Using time-lapse imaging, we showed that mibolerone influences axonal transport in a time- and dose-dependent manner. Mibolerone induces a suppression of anterograde transport at a short time-scale (hours), while enhancing it at a longer time-scale (days). We postulate that androgens may affect anterograde transport differently depending on the activation of membrane-based or intracellular androgen receptors that control, respectively, stability and expression of motor proteins. These findings are relevant for understanding the role of androgens in axonal transport and its fluctuations during puberty.