Objective To observe the retinal nerve fiber layer (RNFL) thickness and optic disc morphology in guinea pigs by optical coherence tomography (OCT), and to investigate the correlation of spherical equivalent and axial length with those parameters. Methods Twenty healthy guinea pigs were used in this study. Spherical equivalent and axial length of nine guinea pigs were measured, and retinal nerve fiber layer thickness and optic disc morphology in the guinea pigs were analyzed by OCT. Results Spherical equivalent of the guinea pigs was positively correlated with average RNFL thickness, and with superior, temporal, inferior and nasal RNFL thicknesses; while ocular axial length was negatively correlated with average RNFL thickness, and with superior, temporal, inferior and nasal RNFL thicknesses. Spherical equivalent and axial length did not correlate significantly with disc edge area, optic disc area, average cup disc ratio, or cup volume. Vertical cup disc ratio significantly correlated with axial length, but did not with spherical equivalent. Conclusions The retinal nerve fiber layer thickness in guinea pigs is affected by both spherical equivalent and axial length. When guinea pigs are used as an animal model of glaucoma, the effect of refractive state and axial length should be considered.