The purpose of this study was to investigate the effects of surface slope and body posture (i.e., seated and standing) on lower extremity joint kinetics during cycling. Fourteen participants cycled at 250 watts power in three cycling conditions: level seated, uphill seated and uphill standing at a 14% slope. A motion analysis system and custom instrumented pedal were used to collect the data of fifteen consecutive cycles of kinematics and pedal reaction force. One crank cycle was equally divided into four phases (90° for each phase). A two-factor repeated measures MANOVA was used to examine the effects of the slope and posture on the selected variables. Results showed that both slope and posture influenced joint moments and mechanical work in the hip, knee and ankle joints (p < 0.05). Specifically, the relative contribution of the knee joint to the total mechanical work increased when the body posture changed from a seated position to a standing position. In conclusion, both surface slope and body posture significantly influenced the lower extremity joint kinetics during cycling. Besides the hip joint, the knee joint also played the role as the power source during uphill standing cycling in the early downstroke phase. Therefore, adopting a standing posture for more power output during uphill cycling is recommended, but not for long periods, in view of the risk of knee injury.