Arboreal habitats pose several challenges for locomotion resulting from narrow cylindrical surfaces, steep slopes, and secondary branches that can form obstructions. We used laboratory trials to test whether different diameters, slopes, or complexity of branches affected maximum speeds and perch choice of the semi-arboreal white-footed mouse (Peromyscus leucopus). We tested locomotor performance of mice running horizontally and up and down 45° slopes for cylindrical artificial branches with five diameters ranging from 10 to 116 mm and on a subset of diameters for cylinders that were horizontal and had pegs (e.g., secondary branches) every 10 or 20 cm. Slope, diameter, and presence of pegs on top of cylinders had significant and interactive effects on locomotor performance. On horizontal cylinders the speed of mice increased with increased diameter among the three smallest diameters, but changed little with further increases in diameter, whereas for sloped surfaces the extreme diameters had lower speeds than an intermediate diameter. For a given diameter, the speeds of mice were usually faster when running horizontally rather than running uphill or downhill. The presence of pegs greatly decreased running speed compared to unobstructed surfaces, but the magnitude of this effect decreased as diameter increased. The difficulties of maintaining balance and avoiding toppling may have caused much of the decrease in speed and associated increased amounts of pausing. Only 1 of 11 choice tests detected a significant bias of mice favoring the perch that maximized locomotor performance. J. Exp. Zool. 317A:540–551, 2012. © 2012 Wiley Periodicals, Inc.