Hematopoietic stem cell (HSC)-based gene therapy targeting CCR5 represents a promising way to cure human immunodeficiency virus type 1 (HIV-1) infection. Yet the preclinical animal model with transplantation of autologous CCR5-ablated HSCs remains to be optimized. In this study, four Chinese rhesus macaques of simian immunodeficiency virus (SIV) chronic infection were given long-term antiretroviral therapy (ART), during which peripheral CD34⁺ hematopoietic stem and progenitor cells (HSPCs) were purified and infected with CCR5-specific CRISPR/Cas9 lentivirus (three monkeys) or GFP lentivirus (one monkey). After non-myeloablative conditioning, the CCR5-modified or GFP-labeled HSPCs were autotransplanted to four recipients, and ART was withdrawn following engraftment. All of the recipients survived the process of transplantation. The purified CD34⁺ HSPCs harbored an undetectable level of integrated SIV DNA. The efficiency of CCR5 disruption in HSPCs ranges from 6.5% to 15.6%. Animals experienced a comparable level of hematopoietic reconstuction and displayed a similar physiological homeostasis Despite the low-level editing of CCR5 in vivo (0.3%–1%), the CCR5-disrupted cells in peripheral CD4⁺ Effector Memory T cell (TEM) subsets were enriched 2- to 3-fold after cessation of ART. Moreover, two of the three treated monkeys displayed a delayed viral rebound and a moderately recovered immune function 6 months after ART withdrawal. This study highlights the importance of improving the CCR5-editing efficacy and augmenting the virus-specific immunity for effective treatment of HIV-1 infection.