Effects of knee alignment on the internal knee abduction moment (KAM) in walking have been widely studied. The KAM is closely associated with the development of medial knee osteoarthritis. Despite the importance of knee alignment, no studies have explored its effects on knee frontal plane biomechanics during stationary cycling. The purpose of this study was to examine the effects of knee alignment and use of a toe clip on the knee frontal plane biomechanics during stationary cycling. A total of 32 participants (11 varus, 11 neutral, and 10 valgus alignment) performed five trials in each of six cycling conditions: pedaling at 80 rpm and 0.5 kg (40 Watts), 1.0 kg (78 Watts), and 1.5 kg (117 Watts) with and without a toe clip. A motion analysis system and a customized instrumented pedal were used to collect 3D kinematic and kinetic data. A 3 × 2 × 3 (group × toe clip × workload) mixed design ANOVA was used for statistical analysis (p < 0.05). There were two different knee frontal plane loading patterns, internal abduction and adduction moment, which were affected by knee alignment type. The knee adduction angle was 12.2° greater in the varus group compared to the valgus group (p = 0.001), yet no difference was found for KAM among groups. Wearing a toe clip increased the knee adduction angle by 0.95º (p = 0.005). The findings of this study indicate that stationary cycling may be a safe exercise prescription for people with knee malalignments. In addition, using a toe clip may not have any negative effects on knee joints during stationary cycling.