SummaryCPSF100 is a core component of the cleavage and polyadenylation specificity factor (CPSF) complex for 3′‐end formation of mRNA, but it still has no clear functional assignment. CPSF100 was reported to play a role in RNA silencing and promote flowering in Arabidopsis. However, the molecular mechanisms underlying these phenomena are not fully understood. Our genetics analyses indicate that plants with a hypomorphic mutant of CPSF100 (esp5) show defects in embryogenesis, reduced seed production or altered root morphology. To unravel this puzzle, we employed a poly(A) tag sequencing protocol and uncovered a different poly(A) profile in esp5. This transcriptome‐wide analysis revealed alternative polyadenylation of thousands of genes, most of which result in transcriptional read‐through in protein‐coding genes. AtCPSF100 also affects poly(A) signal recognition on the far‐upstream elements; in particular it prefers less U‐rich sequences. Importantly, AtCPSF100 was found to exert its functions through the change of poly(A) sites on genes encoding binding proteins, such as nucleotide‐binding, RNA‐binding and poly(U)‐binding proteins. In addition, through its interaction with RNA Polymerase II C‐terminal domain (CTD) and affecting the expression level of CTD phosphatase‐like 3 (CPL3), AtCPSF100 is shown to potentially ensure transcriptional termination by dephosphorylation of Ser2 on the CTD. These data suggest a key role for CPSF100 in locating poly(A) sites and affecting transcription termination.