Objective To measure the rabbit retinal nerve fiber layer(RNFL) thickness in vivo under different intraocular pressures (IOP) and at different time intervals after acute high IOP, and to obtain the regularity on change of RNFL thickness with acute high IOP. Methods Four groups of acute high IOP model were formed by perfusing the saline water into the anterior chamber of rabbit eyes, then RNFL thickness under different IOPs was measured by optical coherence tomography with radial scanning mode whose center was optical papilla and diameter was 6 mm. Results The absolute change values of RNFL thickness were(-27.16±14.24), (-33.33±6.74), (-48.75±5.24), (-67.29±3.89) μm under different IOPs of (4.50±0.35), (6.07±0.31), (7.74±0.26), (10.71±0.07) kPa, respectively. The linear relationship was found between the relative change of RNFL thickness and IOP. IOP could return to the normal level within two days after acute high IOP, while the RNFL thickness could be restored at the second week, and it was easier to restore if the IOP was lower than 6.65 kPa. Conclusions The acute high IOP could cause RNFL thickness to decrease significantly with the increased acute high IOP, but it will be restored after some time.