Healthcare workers (HCWs) are at increased risk of latent tuberculosis (TB) infection (LTBI) and TB disease.We conducted an updated systematic review of the prevalence and incidence of LTBI in HCWs in low- and middle-income countries (LMICs), associated factors, and infection control practices. We searched MEDLINE, Embase and Web of Science (January 1, 2005–June 20, 2017) for studies published in any language. We obtained pooled estimates using random effects methods and investigated heterogeneity using meta-regression.85 studies (32 630 subjects) were included from 26 LMICs. Prevalence of a positive tuberculin skin test (TST) was 14–98% (mean 49%); prevalence of a positive interferon-γ release assay (IGRA) was 9–86% (mean 39%). Countries with TB incidence ≥300 per 100 000 had the highest prevalence (TST: pooled estimate 55%, 95% CI 41–69%; IGRA: pooled estimate 56%, 95% CI 39–73%). Annual incidence estimated from the TST was 1–38% (mean 17%); annual incidence estimated from the IGRA was 10–30% (mean 18%). The prevalence and incidence of a positive test was associated with years of work, work location, TB contact and job category. Only 15 studies reported on infection control measures in healthcare facilities, with limited implementation.HCWs in LMICs in high TB incidence settings remain at increased risk of acquiring LTBI. There is an urgent need for robust implementation of infection control measures.