The importance of U1 snRNA binding free energy in the regulation of alternative splicing has been studied in some genes with site-directed mutagenesis. Here we report a large-scale analysis of its impact on 50 splice site (50ss) selection in human genome. The results show that free energy exerts different effects on alternative 50ss choice in different situations and 8.1 kcal/mol is a hreshold. When both free energies of two competing 50ss are larger than 8.1 kcal/mol, the 50ss with lower free energy is more frequently used. However, in other pairs of 50ss, lower-free-energy 50ss does not seem to be favored and even the other 50ss is used more frequently, which suggests that very low binding free energy would impair splicing. Some observations hold true only for those alternative 50 splicing with short alternative exons (<50nt), which implies a complex mechanism of 50ss selection involving both U1 snRNA binding free energy and regulatory factors.