The regulation of splice site (SS) usage is important for alternative pre-mRNA splicing and thus proper expression of protein isoforms in cells; its disruption causes diseases. In recent years, an increasing number of novel regulatory elements have been found within or nearby the 3'SS in mammalian genes. The diverse elements recruit a repertoire of trans-acting factors or form secondary structures to regulate 3'SS usage, mostly at the early steps of spliceosome assembly. Their mechanisms of action mainly include: (1) competition between the factors for RNA elements, (2) steric hindrance between the factors, (3) direct interaction between the factors, (4) competition between two splice sites, or (5) local RNA secondary structures or longer range loops, according to the mode of protein/RNA interactions. Beyond the 3'SS, chromatin remodeling/transcription, posttranslational modifications of trans-acting factors and upstream signaling provide further layers of regulation. Evolutionarily, some of the 3'SS elements seem to have emerged in mammalian ancestors. Moreover, other possibilities of regulation such as that by non-coding RNA remain to be explored. It is thus likely that there are more diverse elements/factors and mechanisms that influence the choice of an intron end. The diverse regulation likely contributes to a more complex but refined transcriptome and proteome in mammals.