PURPOSE. The purpose of this study was to characterize the pulsatile motion of trabecular meshwork (TM) in normal subjects and demonstrate its changes in accommodation with phase-sensitive optical coherence tomography (PhS-OCT). METHODS. A new PhS-OCT laboratory prototype was designed to measure pulsatile TM motion in 13 healthy humans. Two sets of images were captured in 10 subjects, first with best corrective refraction and the other with an additional 3.0 diopters of accommodation. In each image, both maximum velocity (MV) and cumulative displacement (CD) in two selected regions of TM, the internal (IMV and ICD) and external (EMV and ECD) region, were measured. RESULTS. For all parameters the intraclass correlation coefficient was > 0.75. Neither MV nor CD was significantly different between eyes in individual subjects (PIMV = 0.967, PEMV = 0.391, PICD = 0.603, PECD = 0.482). In 26 eyes, with best corrective refraction, the EMV was higher than the IMV (23.9 6 9.8 vs. 18.9 6 8.08 lm/s; P = 0.0001), as was the ECD compared with the ICD (0.340 60.125 vs. 0.264 6 0.111 lm; P = 0.000004). With accommodation, MV and CD significantly increased (PIMV = 0.0003, PEMV = 0.0003, PICD = 0.019, and PECD = 0.007), whereas MV and CD in the external region were still larger than those in the internal area (PEMV vs. IMV = 0.009, PECD vs. ICD = 0.023). CONCLUSIONS. This study demonstrates the differences in TM motion between the internal and external regions of TM and displays its change with accommodation. The findings and good reproducibility suggest PhS-OCT helps to understand TM function in regulation of IOP, and, with further refinements, it may be useful in clinical management of glaucoma.