Objective: Investigating the implementation of labor protection regulations in gestation for working women in Hunan province, and analysing its influence on the fertility desire for two-child. Methods: By stratified cluster random sampling, the questionnaire survey was conducted among working women who are childbearing age. Results: The implementation of labor protection regulations was different among enterprises. And the state-owned enterprises relatively did better in the implementation of maternity leave, hours of labour and caring for labor. Nursing leave, one hour time for lactation, adaptability to the original labor and maternal care measures for aged significantly influence working women’s fertility desire for two-child. Conclusion: The relevant government departments should ensure good implementation of labor protection regulations and improve constantly, in order to increase the compatibility between work and fertility of working women and so as to promote the realization of the new childbearing policy