The transverse carpal ligament (TCL) is a component of the flexor pulley system of the wrist, keeping the flexor tendons in place by resisting their volar displacement. The purpose of this study was to investigate the in vivo biomechanical interaction between the TCL and flexor tendons in response to tendon tensioning with the wrist at various postures. In eight healthy subjects, the flexor digitorum superficialis and profundus tendons were tensioned by isometrically applying loads (5, 10, and 15 N) to the index finger while the wrist posture was at 20° extension, neutral, 20° flexion, and 40° flexion. The TCL and flexor tendons were imaged at the distal carpal tunnel cross section using ultrasound. The volar-dorsal positions of the tendons, TCL arch height, and TCL-tendon distances were calculated. With increasing wrist flexion, the flexor tendons moved volarly, the TCL arch height increased, and the TCL-tendon distances decreased, indicating that the flexor tendons contacted the TCL and pushed it volarly. The TCL-tendon interaction was amplified by the combination of finger loading and wrist flexion. This study provides in vivo evidence of the biomechanical interaction between the TCL and flexor tendons. Repetitive TCL-tendon interactions may implicate the interacting tissues and the median nerve resulting in tissue maladaptation and nerve compression.