Accurate segregation of the genetic material during cell division requires that sister chromatids are kept together by cohesion proteins until anaphase, when the chromatids become separated and distributed to the two daughter cells. Studies in yeast revealed that chromatid cohesion is essential for viability and is triggered by the conserved protein Eco1 (Ctf7). Cohesion must be established already in S phase in order to tie up sister chromatids instantly after replication, but how this crucial timing is achieved remains enigmatic. Here, we report that in yeast and humans Eco1 is directly physically coupled to the replication protein PCNA, a ring-shaped cofactor of DNA polymerases. Binding to PCNA is crucial, as yeast Eco1 mutants deficient in Eco1-PCNA interaction are defective in cohesion and inviable. Our study thus indicates that PCNA, a central matchmaker for replication-linked functions, is also crucially involved in the establishment of cohesion in S phase.