The development of an effective T cell based HIV vaccine would need to elicit cell mediated immune responses with superior magnitude, breadth, and quality. Since blocking the interactions between inhibitory receptors with their associated ligands using soluble PD-1 (sPD-1) and soluble Tim-3 (sTim-3) have been shown to reverse T cell exhaustion and enhance cell mediated immune responses, we tested if co-administration of sPD-1 and sTim-3 with an adenovirus vectored SIV vaccine (rAd5-SIV) can enhance cell mediated immune responses. The frequency of SIV antigen specific IFN-γ spot-forming cells and the secretion of IFN-γ and TNF-α by splenocytes from rAd5-SIV immunized mice was significantly increased when stimulated ex vivo with SIV peptides in the presence of sPD-1 or sTim-3 or both sPD-1 and sTim-3. The magnitude of cell mediated immune responses elicited by rAd5-SIV was enhanced by co-administration of sPD-1 and sTim-3. Co-administration of both sPD-1 and sTim-3 induced higher frequency of SIV antigen specific IFN-γ⁺ spot-forming cells to poorly immunogenic Vif and Tat. The percentage of cell mediated responses for each SIV antigen became more balanced, with reduction to Gag but induction to non-structural proteins. Furthermore, co-injection of rAd5-sPD1 and rAd5-sTim3 with rAd5-SIV in mice enhanced T cell proliferation capability and generated more antigen specific IFN-γ⁺ CD4⁺ and CD8⁺ T cells. Our study provided a new approach to enhance vaccine induced cell mediated immune responses, which may be applicable to improve the efficacy of vaccines against SIV/HIV.