Hydrogen sulfide (H2S), a traditionally known cytotoxic gas, has been recently included in the gasotransmitters family. Previous studies demonstrated that lifelong treatment with a slow H2S releasing donor extends yeast chronological lifespan (CLS), but it is not clear when the action of H2S benefits to CLS during yeast growth. Therefore, we investigated the effects of short H2S treatments at different time during yeast cell growth in the lifespan extension by using NaHS, a fast H2S-releasing donor. We show that short NaHS treatments at 4 days after inoculation extend yeast CLS while NaHS treatments earlier than 3 days after inoculation fail to do so. To reveal the mechanism of different consequences on yeast CLS of NaHS treatments at different times, we analyzed the transcriptome of Saccharomyces cerevisiae strain BY4742 with or without the early and late NaHS treatments. We found that the early and late NaHS treatments had similar effects on the expression of genes involved in pathways which are related to CLS regulation. Follow up qPCR and ROS analyses suggest that altered expression of some antioxidant genes by the early NaHS treatments were not stable enough to benefit CLS. Moreover, transcriptome data also indicated that some genes were regulated differently by the early and late H2S treatment such as genes involved in cell wall integrity. Specifically, we found that the expression of YPK2, a human SGK2 homolog and also a key regulator of the yeast cell wall synthesis, was significantly altered by the late NaHS treatment but not altered by the early NaHS treatment. Finally, the key role of YPK2 in CLS regulation by H2S is revealed by CLS data showing that the late NaHS treatment did not enhance the CLS of a ypk2 knockout mutant. This study sheds light on the molecular mechanism of CLS extension induced by H2S, and for the first time addresses the importance of H2S treatment timing for lifespan extension. Overall design: Four samples, three replicates each. Names are Early_control, Early_NaHS_treatment, Late_control and Late_NaHS_treatment.