Objective: To investigate the effect of inhibiting accommodation on ocular refractive development of guinea pigs in different monochromatic lights. Methods: Twenty-four pigmented guinea pigs were randomly divided into three groups with 8 animals per group: short-wavelength light (SL, 430 nm) group, middle-wavelength light (ML,530 nm) group and broad-band light (BL, 5 000 K color temperature) group. The right eyes of all animals were treated by 1% Atropine solution once a day for 6 weeks. Measurements of ocular refraction, corneal curvature, and axial length were performed at the start and the end of the study. Results: There was no significant difference in bilateral ocular refraction for all groups at the beginning of the experiment (about 4.25 D, P>0.05) and in ipsilateral ocular refraction among groups at the start of the experiment (P>0.05). But at the end of the experiment,significant differences were detected between binocular refraction of the ML group (P=0.028) and the SL group (P=0.0003), however, there was no significant difference between bilateral refraction in the BL group (P=0.7486).There were significant differences in refraction between the left eyes of any two groups (P<0.05), between the right eyes of the ML and BL group (P=0.001), and between the right eyes of the ML and SL (P<0.001) at 6 weeks.No significant refractive difference was detected between the right eyes of the SL and BL groups (P=0.072). The vitreous length was about 3.2 mm in bilateral eyes of all groups at the onset of the experiment (all inner- or inter-group P>0.05). After the experiment, the bilateral difference in vitreous length was significant in the ML group(P=0.0113) and the SL group (P=0.0017), but not significant in the BL group (P=0.9371). There were significant vitreous differences in right or left eyes among the groups at the end of the experiment (P<0.01). There were no significant inter-group (ipsilateral) or inner-group (bilateral) differences at any time in any of corneal radius of curvature, anterior segment length and lens thickness (P>0.05 for all comparisons). Conclusion: 1% atropine can strengthen the effect of vitreous elongation and myopic formation on guinea pig eyes in 530 nm monochromatic light. Moreover, atropine can weaken the effect of vitreous shortening and hyperopic formation on guinea pig eyes in 430 nm monochromatic light. Ocular accommodation response should involve in the mechanism of refractive development of guinea pig in monochromatic light. Atropine can influence the refractive development of guinea pig in monochromatic light possibly by inhibiting accommodation response.