Background: Despite demonstration of molecular and biochemical changes induced by heat shock on Leishmania, the immunological importance of such changes has not been elucidated. Objective: Studying the effect of two excretory factors prepared under heat shock and ambient temperature from Leishmania major on Balb/c splenocytes function. Methods: The parasites were cultured at 25°C and then subjected to heat shock by overnight incubating at 35°C. Both 25°C (R25) and 35°C (R35) supernatants were collected and used for biochemical analysis and as culture media for T cell activation. The protein profile of two supernatants was studied by SDS-PAGE. Splenocytes of Balb/c mice were stimulated by concavalin-A in the presence of RPMI, R25 and R35. Consequently, CD25 expression was studied by means of flow cytometry and the production of IFN-γ and IL-4 was determined by ELISA. Results: SDS-PAGE analysis showed distinct protein profile for R25 and R35. Significant decrease in IFN-γ and IL-4 production by cells cultured in R35 was noticed (p<0.001). CD25 expression was also down-regulated (p<0.01). In contrast, cytokine production in the supernatants of the cells cultured in R25 demonstrated suppression on IL-4 (p<0.01) but not on IFN-γ production. In parallel, CD25 expression was not down-regulated in the presence of R25 medium. Conclusion: The data suggest that heat shock-induced excretory factor of L. major is capable of inhibiting T cell function by decreasing IFN-γ production and down-regulating CD-25 expression which might be involved in the induction of immunosuppression in this susceptible murine strain. Furthermore, we suppose that the supernatant of L. major culture in ambient temperature have an intrinsic ability to promote Th1 response.