Currently, circadian regulation of immune molecules in lower vertebrates, particularly, diurnal oscillation in the immune status of a fish, is not well understood. In this study, the diurnal oscillation of toll-like receptor (Tlr) 9, which plays a role in pathogen recognition, was investigated in the Japanese medaka fish (Oryzias latipes). We confirmed the expression of tlr9 and clock genes (bmal1 and clock1) in the central and peripheral tissues of medaka. These genes were expressed in a diurnal manner in medaka acclimated to a 12-h:12-h light-dark (12:12 LD) cycle. In addition, increased tlr9 expression was detected in medaka embryo cells (OLHdrR-e3) overexpressing both bmal1 and clock1 genes; however, this result was not obtained when only one or neither of the genes was overexpressed. This suggests that the increase in expression was mediated by the Bmal1 and Clock1 proteins together. In vitro stimulation of the head kidney with CpG-oligodeoxynucleotides (CpG-ODNs) at different zeitgeber times (ZTs; ZT0 = light on, ZT12 = light off) affected the degree of tlr9 gene expression, showing high and low responsiveness to CpG-ODN stimulation at ZT6/10 and ZT18/22, respectively. Similarly, bacterial infection at different ZT points induced a difference in the expression of Tlr9 signaling pathway-related genes (tlr9 and myd88). These results suggested that fish tlr9 exhibits diurnal oscillation, which is regulated by clock proteins, and its responsiveness to immune-stimulation/pathogen infection depends on the time of the day.