This study evaluated the effects of sludge lysate (SL) on the anaerobic bioreduction of Cr(VI) and the role of sludge humic acid (SHA) during this process. The results showed that supplement of SL significantly enhanced the efficiency of Cr(VI) bioreduction by 29.61%, in 12 h compared with that of the control without SL. Moreover, SHA exhibited promoting effects on bioreduction of Cr(VI), and the promotion increased with increasing SHA concentrations from 100 to 300 mg/L. In the presence of 300 mg/L SHA, Cr(VI) (98.21 mg/L) was completely reduced after 24 h with a removal rate increased by 34.3% compared with that of the control without SHA. Further investigation on the bioaugmentation mechanism of SHA by studying the nature of SHA and the reaction mechanism between SHA and Cr(VI) revealed that SHA exhibited a strong adsorption ability, which could adsorb and combine with Cr(VI). The adsorption capacity of Cr(VI) by SHA was calculated as 34.4 mg/g with 0.2 g of SHA and 10 mg/L of Cr(VI). It could also act as redox mediators to accelerate the electron transfer between microorganisms and Cr(VI) to promote reduction of Cr(VI). Furthermore, the effects of SL on the microbial community compositions of the anaerobic Cr(VI) bioreduction system were studied. Brachymonas was the primary bacteria at the genus level. The abundance of electroactive bacteria, such as Acinetobacter, Pseudomonas, and Arcobacter, increased in the SL-amended system. These findings expand the versatility of SL and justify wider use of residual activated sludge, which might contribute to the treatment of heavy metal-contaminated wastewater.