BK and JC Polyomavirus are closely related and establish persistence in infected subjects. Recent studies suggest that JC Polyomavirus replication prevents BK Polyomavirus-related pathologies in kidney transplant recipients. One potential mechanism of this competition could involve viral microRNAs cross-reacting, as they are highly homologous between species. In fact, bkv-miR-B1-3p is strictly identical to jcv-miR-J1-3p, whereas species-specific miRNAs bkv-miR-B1-5p and jcv-miR-J1-5p differ barely. Early detection of jcv-miR-J1-5p in urine significantly reduces the risk of BK Polyomavirus DNAemia in kidney transplant recipients in a case-control study including 39 patients (odds ratio [95 % Confidence Interval] = 0.00 [0.00-0.65], p = 0.012). In vitro modeling revealed that prior infection with JC Polyomavirus reduces the ability of BK Polyomavirus to grow in immortalized human renal proximal tubular epithelial cells, without significant expression of JC Polyomavirus proteins. The JC Polyomavirus-specific miRNA jcv-miR-J1-5p was discovered to decrease BK Polyomavirus TAg mRNA expression, without affecting early genome replication, like the known regulatory effect of BK Polyomavirus-encoded bkv-miR-B1-3p and bkv-miR-B1-5p. An archetypal strain of JC Polyomavirus engineered to quench miRNA maturation did not inhibit BK Polyomavirus infection, unlike the wild-type strain, confirming that the inhibitory effect of JC Polyomavirus is due to miRNAs. These results suggest that JC Polyomavirus-specific miRNA jcv-miR-J1-5p limits BK Polyomavirus infectivity and early TAg expression, with an intensity similar to BK Polyomavirus miRNAs. This mechanism might explain in vivo competition for viral replication between BK Polyomavirus and JC Polyomavirus infections.