Background/Aims: Little is known regarding the activation of knee and hip muscles during backward walking in patellofemoral pain syndrome. This study examineD the effects of backward walking and forward walking on the activation of knee extensors, hip abductors, and adductors in patients with patellofemoral pain syndrome. Methods: A total of 20 females with patellofemoral pain syndrome and 20 age-matched typically healthy female controls participated in this study. Surface electromyography from vastus medialis obliquus, vastus lateralis, gluteus medius, and adductor longus muscles were collected during forward walking and backward walking. Findings: The patellofemoral pain syndrome group had a significantly higher normalised root mean square of the vastus medialis obliquus, vastus lateralis and gluteus medius muscles (P=0.001), without significant difference in adductor longus muscle activity during backward walking versus forward walking (P=0.098). During forward walking, the patellofemoral pain syndrome group showed significantly higher activation of adductor longus muscle (P=0.001) and significantly lower activation of the gluteus medius muscle (P=0.002) compared to the healthy group. During backward walking there was a significant increase in the vastus medialis obliquus and adductor longus muscle activity of the patellofemoral pain syndrome group compared to the control group (P=0.003, 0.001) respectively. Conclusions: Clinicians should consider backward walking training to increase the muscle strength of knee extensors and hip abductors when developing rehabilitation programmes for patients with patellofemoral pain syndrome.