The optimal timing for initiating renal replacement therapy (RRT) in patients with acute kidney injury (AKI) remains controversial. We conducted a meta-analysis with trial sequential analysis (TSA) of randomized controlled trials (RCTs) using PUBMED, Cochrane Library databases, and Web of Science (from January 1, 1985, to August 21, 2016). Adult patients with AKI who received RRT with different timing were included. The primary outcome was mortality. The secondary outcomes were intensive care unit (ICU) length of stay (LOS) and hospital LOS. We included 9 RCTs with a total of 1636 participants. No differences between the early RRT group and the late RRT group were found with respect to mortality (38% vs 41.4%; relative risk, 0.93; 95% confidence interval CI, 0.74-1.18). However, TSA showed that the cumulative Z-curve did not cross either the conventional boundary for benefit or the trial sequential monitoring boundary, indicating insufficient evidence. Similarity, there were no findings of benefits in terms of reduction in the ICU LOS (standard difference in the means, -0.32 days; 95% CI, -0.71 to 0.07 days) and hospital LOS (standard difference in the means, -1.11 days; 95% CI, -2.28 to 0.06 days). Meanwhile, the results of TSA did not confirm this conclusion. Although conventional meta-analysis showed that early initiation of RRT in patients with AKI was not associated with decreased mortality, ICU LOS and hospital LOS, TSA indicated that the data were far too sparse to make any conclusions. Therefore, well-designed, large RCTs are needed.