Gene amplification is a hallmark of cancer with chromosomal instability although the underlying mechanism by which altered copy numbers are maintained is largely unclear. Cohesin, involved in sister chromatid cohesion, DNA repair, cell cycle progression and transcriptional regulation of key developmental genes, is frequently overexpressed in human cancer. Here we show that cohesin-dependent change in DNA replication controls the copy numbers of amplified genes in cancer cells with chromosomal instability. We found that the down-regulation of elevated cohesin leads to copy number-associated gene expression changes without disturbing chromosomal segregation. Highly amplified genes form typical long-range chromatin interactions, which are stabilized by enriched cohesin. The spatial proximities among cohesin binding sites within amplified genes are decreased by RAD21-knockdown, resulting in the rapid decline of amplified gene expression. After several passages, cohesin depletion inhibits DNA replication initiation by reducing the recruitment of pre-replication complexes such as minichromosome maintenance subunits 7 (MCM7), DNA polymerase α, and CDC45 at replication origins near the amplified regions, and as a result, decreases the DNA copy numbers of highly amplified genes. Collectively, our data demonstrate that cohesin-mediated chromatin organization and DNA replication are important for stabilizing gene amplification in cancer cells with chromosomal instability.