To investigate the effects of spectral composition and light intensity on natural refractive development in guinea pigs. A total of 124 pigmented guinea pigs (2-week-old) were randomly assigned to three groups at high (Hi; 4000 lx), medium (Me; 400 lx) and low (Lo; 50 lx) light intensities under a 12:12 light/dark cycle for 6wk. Each group was subdivided into subgroups with the following spectra: broad spectrum Solux halogen light (BS), 600 nm above-filtered continuous spectrum (600F), 530 nm above-filtered continuous spectrum (530F), and 480 nm above-filtered continuous spectrum (480F; HiBS: n=10, Hi600F: n=10, Hi530F: n=10, Hi480F: n=10, MeBS: n=10, Me600F: n=10, Me530F: n=10, Me480F: n=10, LoBS: n=11, Lo600F: n=12, Lo530F: n=10, Lo480F: n=11). Refractive error, corneal curvature radius, and axial dimensions were determined by cycloplegic retinoscopy, photokeratometry, and A-scan ultrasonography before and after 2, 4, and 6wk of treatment. Average changes from both eyes in the ocular parameters and refractive error were compared among different subgroups. After 6wk of exposure, high-intensity lighting enhanced hyperopic shift; medium- and low-intensity lighting enhanced myopic shift (P<0.05). Under the same spectrum, axial increase was larger in the low light intensity group than in the medium and high light intensity groups (HiBS: 0.65±0.02 mm, MeBS: 0.67±0.01 mm, LoBS: 0.82±0.02 mm; Hi600F: 0.64±0.02 mm, Me600F: 0.67±0.01 mm, Lo600F: 0.81±0.01 mm; Hi530F: 0.64±0.02 mm, Me530F: 0.67±0.01 mm, Lo530F: 0.73±0.02 mm; Hi480F: 0.64±0.01 mm, Me480F: 0.66±0.01 mm, Lo480F: 0.72±0.02 mm; P<0.05). Under 400 lx, there was a faster axial increase in the MeBS group than in the Me480F group (P<0.05). Under 50 lx, axial length changes were significantly larger in LoBS and Lo600F than in Lo530F and Lo480F (P<0.01). Under high-intensity lighting, high light intensity rather than spectrum distributions that inhibits axial increase. Under medium- and low-intensity lighting, filtering out the long wavelength inhibits axial growth in juvenile guinea pigs.