Arbuscular mycorrhizal fungi (AMF) have been shown to assist plants in increasing metal tolerance and accumulation in heavy metal (HM)-contaminated soils. Herein, a greenhouse pot experiment was conducted to assess the interactions of growth substrates (S1, S2, and S3, respectively) with various HM contamination and nutrient status sampling from a typical contaminated soil and tailings in Shuikoushan lead/zinc mining in Hunan province, China, and AMF inoculation obtained from plants in uncontaminated areas (Glomus mosseae,Glomus intraradices,and uninoculated, respectively) on the biomass and uptake of HMs and phosphorus (P) by the black locust plant (Robinia pseudoacaciaL.). The results indicated that the inoculation with AMF significantly enhanced the mycorrhizal colonization of plant roots compared with the uninoculated treatments, and the colonization rates were found to be higher in S1 and S2 compared with S3, which were characterized with a higher nutrient availability and lead concentration. The biomass and heights ofR. pseudoacaciawere significantly increased by AMF inoculation in S1 and S2. Furthermore, AMF significantly increased the HM concentrations of the roots in S1 and S2 but decreased the HM concentrations in S3. Shoot HM concentrations varied in response to different AMF species and substrate types. Mycorrhizal colonization was found to be highly correlated with plant P concentrations and biomass in S1 and S2, but not in S3. Moreover, plant biomass was also significantly correlated with plant P concentrations in S1 and S2. Overall, these findings demonstrate the interactions of AMF inoculation and growth substrates on the phytoremediation potential ofR. pseudoacaciaand highlights the need to select optimal AMF isolates for their use in specific substrates for the remediation of HM-contaminated soil.