Background: There is a circadian rhythm in heart rate and conversely, bradyarrhythmias occur at night. Recent work from our lab has demonstrated heart rate rhythmicity to be underscored by a diurnal fluctuation in the key pacemaking channel HCN4, and its corresponding funny current If, likely driven by a local clock transcription factor BMAL1 (1). We have also previously shown that microRNAs (miRs) regulate sinus node pacemaking by direct negative regulation of Hcn4 transcription (2). Here we tested whether rhythmic miR expression explains Hcn4 and Bmal1 rhythmicity in the sinus node. Methods: Sinus node biopsies were collected from nocturnal adult male C57BL/6J mice at zeitgeber time (ZT) 0, ZT6, ZT12 and ZT18 (n=7/8). TaqMan Array MicroRNA A+B Cards were used to measure the expression of 750 miRs and 6 reference transcripts. JTK_Cycle (3) and sine wave fitting were used to identify rhythmic miRs (exhibiting a ~24 h periodicity) and in silico prediction algorithms RNA22, TargetScan and PITA applied to identify potential 3′ untranslated region (3′UTR) target sites. Functionality of predicted sites was tested by dual luciferase reporter gene assay; recombinant plasmids, in which a luciferase coding sequence was fused to (i) Hcn4 3’UTR and (ii) Bmal1 3’UTR, were co-transfected individually with (i) 19 and (ii) 11 miR mimics in H9C2 rat myoblast cells and compared to control plasmid. Firefly luciferase was measured and normalised to Renilla luciferase in 7 independent batches of cells. Results: JTK_Cycle analysis identified 159 rhythmic microRNAs (Bonferroni adjusted P<0.05). Sine wave fitting was carried out on this dataset to identify false positives, after which 56 miRs were found to exhibit day-night rhythms in the sinus node. Of these miRs, 39 and 11, respectively, were expressed antiphase to Hcn4 and Bmal1, indicating potential for negative regulation. Bioinformatics analyses identified 19 miRs with binding sites on the Hcn4 3’UTR whereas 11 miRs were predicted for Bmal1 3’UTR binding. Compared to control, 6/19 miRs predicted to target Hcn4, and 2/11 miRs predicted to target Bmal1, resulted in a significant reduction in reporter bioluminescence vs. control (P<0.05, unpaired t test). Compared to co-transfection with negative control C.elegans miR-67, miRs-146a-5p, -128-3p, -150-5p and -203-3p significantly reduced Hcn4 3′UTR activity (P<0.05, unpaired t test). Sine curve analysis of these miRs determined them to be expressed antiphase (peak ~ZT9) to Hcn4 (peak ~ZT21). Conclusions: This is the first report of rhythmic miR expression in the sinus node. miRs-146a-5p, -128-3p, -150-5p and -203-3p are novel regulators of Hcn4 transcription and ongoing studies are examining their physiological relevance for the circadian rhythm in sinus node pacemaking. This could be the first step towards chronotherapeutic targets for the treatment of heart rhythm disturbances.