Hikers and soldiers usually walk up and down slopes with a load carriage, causing injuries of the musculoskeletal system, especially during a prolonged load journey. The slope walking has been reported to lead to higher leg extensor muscle activities and joint moments. However, most of the studies investigated muscle activities or joint moments during slope walking without load carriage or only investigated the joint moment changes and muscle activities with load carriages during level walking. Whether the muscle activation such as the signal amplitude is influenced by the mixed factor of loads and grades and whether the influence of the degrees of loads and grades on different muscles are equal have not yet been investigated. To explore the effects of backpack loads on leg muscle activation during slope walking, ten young male participants walked at 1.11 m/s on a treadmill with different backpack loads (load masses: 0, 10, 20, and 30 kg) during slope walking (grade: 0, 3, 5, and 10°). Leg muscles, including the gluteus maximus (GM), rectus femoris (RF), hamstrings (HA), anterior tibialis (AT), and medial gastrocnemius (GA), were recorded during walking. The hip, knee, and ankle extensor muscle activations increased during the slope walking, and the hip muscles increased most among hip, knee, and ankle muscles (GM and HA increased by 46% to 207% and 110% to 226%, respectively, during walking steeper than 10° across all load masses (GM: p = 1.32 × 10−8 and HA: p = 2.33 × 10−16)). Muscle activation increased pronouncedly with loads, and the knee extensor muscles increased greater than the hip and ankle muscles (RF increased by 104% to 172% with a load mass greater than 30 kg across all grades (RF: p = 8.86 × 10−7)). The results in our study imply that the hip and knee muscles play an important role during slope walking with loads. The hip and knee extension movements during slope walking should be considerably assisted to lower the muscle activations, which will be useful for designing assistant devices, such as exoskeleton robots, to enhance hikers’ and soldiers’ walking abilities.