Antiretroviral therapy is capable of inhibiting HIV replication, but it fails to completely achieve a cure due to HIV persistence. The commonly used HIV cure approach is the “shock and kill” strategy, which employs latency-reversing agents to trigger viral reactivation and boost cellular immunity. Finding the appropriate drug combination for the “shock and kill” strategy would greatly facilitate clinical trials. The toll-like receptor (TLR) 7 agonist GS–9620 and nicotinamide (NAM) are reported as potential latency-reversing agents. Herein, we found the absence of viral reactivation when SHIVSF162P3-aviremic rhesus macaques were treated with GS–9620 monotherapy. However, our findings demonstrate that viral blips emerged in half of the macaques treated with the combination therapy of GS–9620 and NAM. Notably, an increase in the reactivation of the replication-competent latent virus was measured in monkeys treated with the combination therapy. These findings suggest that the GS–9620 and NAM combination could be used as a multipronged HIV latency stimulation approach, with potential for optimizing antiviral therapy design.