Nitric oxide (NO) plays many roles in the immune system. It has been known that NO rescues thymocytes from glucocorticoid (GC)-induced apoptosis. However, the downstream target of NO in the protection from GC-induced thymocyte apoptosis has yet to be identified. We previously reported that GC sensitivity of developing thymocytes is dependent on the expression level of SRG3. In the present report, we found that NO repressed the SRG3 expression in both primary thymocytes and 16610D9 thymoma cells. Specifically, NO down-regulated the transcription of SRG3 via the inactivation of the transcription factor Sp1 DNA-binding activity to the SRG3 promoter. In addition, overexpression of SRG3 by a heterologous promoter reduced NO-mediated rescue of thymocytes from GC-induced apoptosis. These observations strongly suggest that NO may be involved in protecting immature thymocytes from GC-induced apoptosis by repressing the SRG3 expression in thymus.