Non-coding Y RNAs have recently been identified as essential novel factors for chromosomal DNA replication in mammalian cell nuclei, but mechanistic details of their function have not been defined. Here, we identify the execution point for Y RNA function during chromosomal DNA replication in a mammalian cell-free system. We determined the effect of degradation of Y3 RNA on replication origin activation and on fork progression rates at single-molecule resolution by DNA combing and nascent-strand analysis. Degradation of Y3 RNA inhibits the establishment of new DNA replication forks at the G1- to S-phase transition and during S phase. This inhibition is negated by addition of exogenous Y1 RNA. By contrast, progression rates of DNA replication forks are not affected by degradation of Y3 RNA or supplementation with exogenous Y1 RNA. These data indicate that Y RNAs are required for the establishment, but not for the elongation, of chromosomal DNA replication forks in mammalian cell nuclei. We conclude that the execution point for non-coding Y RNA function is the activation of chromosomal DNA replication origins.