Introduction: Pain and overuse injuries of the knee joint is prevalent among cyclists. The bicycle adjustment in accordance with the cyclist’s body mechanics is a common way to reduce the risk of overuse injuries. The aim of this study is to investigate the effect of changes in bicycle pedal width on the kinematics of segments and joints of lower extremity and its association with the risk of knee overuse injuries during pedaling. Materials and Methods: 10 professional cyclists of Shiraz City, Iran, pedaled at 100% of maximum power output with four different pedal widths (Q0: conventional pedal width, Q1: Q0 + 1cm, Q2: Q0 + 2cm, and Q3: Q0 + 3cm). The angle of the lower extremity segments and joints was recorded three dimensionally for thirty seconds during pedaling in each pedal width by myoMotion system. The minimum, maximum, and mean angles and range of motion (ROM) variables of hip and ankle (sagittal plane), knee joint (sagittal and frontal planes), and thigh and shank angles in the frontal plane were calculated. One-way repeated measures analysis of variance (ANOVA) and Bonferroni post-hoc test were used to identify significant changes. Results: The statistical results showed that changes in the pedal width had a significant effect of on minimum (P = 0.035), maximum (P ≤ 0.042), and mean (P ≤ 0.020) of shank abduction/adduction and minimum (P = 0.015), mean (P ≤ 0.022), and ROM (P ≤ 0.018) of ankle dorsiflexion/plantar flexion, while changes in the pedal width had no significant effect on other kinematics parameters. Conclusion: The results indicate that pedal width of Q1 has the highest potential to lower the risk of knee injury and provide increased efficiency whilst cycling; Still, the standard pedal width of road bikes (Q0) seemed not appropriate for professional Iranian cyclists since it increases the risk of knee joint overuse injuries.