As the smallest living primate, the mouse lemur is a suitable model for reconstructing the locomotor mechanisms by which primate ancestors might have responded to the challenges of an arboreal environment. In this study, we tested the effects of substrate diameter and orientation on quadrupedal gait kinematics in mouse lemurs (Microcebus murinus). Mouse lemurs highly preferred asymmetrical to symmetrical gaits as they moved across a flat board and poles of three diameters (2.5, 1.0, and 0.5 cm), set at horizontal, 30°inclined, and 30°declined orientations. During symmetrical gaits, mouse lemurs used diagonal sequence walking and ambling gaits on the same substrates and at the same duty factors for which some similarly sized nonprimate mammals use lateral sequence gaits, suggesting that reliance on diagonal sequence walking in primates may not be explicitly a response to body size relative to substrate diameter. When using asymmetrical gaits, kinematic adjustments to small diameter and/or nonhorizontal substrates included a preference for transverse gallops over other gaits, the avoidance of whole-body suspensions, increases in limb contact duration, and increases in the time interval between the landing of trailing and leading limbs. All of these adjustments are consistent with increasing locomotor stability by dampening center of mass movements and reducing the forces imparted to the substrate. Like mouse lemurs, small-bodied ancestral primates likely used symmetrical gaits occasionally, but more frequently used asymmetrical gaits that were adjusted in response to challenging substrates. Therefore, asymmetrical gait dynamics should be incorporated into hypotheses addressing early primate locomotor evolution.