The purpose of this study was to investigate the effect of blue light phototherapy on the expression of circadian genes in peripheral blood mononuclear cells (PBMC) and plasma melatonin levels in neonates. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was used to determine the expression of Bmal1 and Cry1 in PBMC, and an enzyme-linked immunosorbent assay was used to determine plasma melatonin levels in 32 breast-milk jaundiced neonates before and after phototherapy, compared with 29 control neonates. The results showed that the expression of Bmal1 was decreased and Cry1 increased significantly after phototherapy. Plasma melatonin levels were decreased after phototherapy. There was no statistical difference in Bmal1 and Cry1 gene expression and plasma melatonin levels in the control group. In conclusion, phototherapy does affect the expression of the circadian genes Bmal1 and Cry1 in PBMC and plasma melatonin concentration in jaundiced neonates. Our results suggest that phototherapy should be timed according to circadian rhythms when treating jaundiced neonates.