Zidovudine (ZDV) is a widely used component of antiretroviral therapy (ART) in resource-limited settings, despite its known adverse effects, which include mitochondrial toxicity in muscle, liver and adipose tissue. It has also been associated with impaired immunological recovery. We hypothesised that ZDV might impair mitochondrial health and survival of primary T cells. We performed a cross-sectional analysis of mitochondrial function, mitophagy and susceptibility to apoptosis in healthy donor primary T cells after exposure to ZDV in vitro, together with T cells from patients who were virologically suppressed on ZDV-containing ART regimens for ≥1 year and age-matched subjects receiving non-ZDV ART regimens. The proportion of T cells expressing mitochondrial reactive oxygen species (mtROS) was significantly higher after in vitro (CD4(+) T cells and CD8(+) T cells) and in vivo (CD4(+) T cells) exposure to ZDV than other antiretroviral agents. We did not detect any effect of ZDV on mitophagy, as indicated by change in autophagic flux. However, spontaneous apoptosis, indicated by upregulation of caspase-3 was greater in ZDV-exposed T cells. In conclusion, ZDV exposure was associated with impaired mitochondrial turnover and increased susceptibility to apoptosis in T cells. These mechanisms could contribute to sub-optimal immune reconstitution.