Abscisic acid (ABA) is a key phytohormone underlying plant resistance to toxic metals. However, regulatory effects of ABA on apoplastic transport in roots and consequences for uptake of toxic metals are poorly understood. Here, we demonstrate how ABA regulates development of apoplastic barriers in roots of two ecotypes of Sedum alfredii and assess effects on cadmium (Cd) uptake. Under Cd treatment, increased endogenous ABA level was detected in roots of non-hyperaccumulating ecotype (NHE) due to up-regulated expressions of ABA biosynthesis genes (SaABA2 and SaNCED) but no change was observed in hyperaccumulating ecotype (HE). Simultaneously, endodermal Casparian strips (CSs) and suberin lamellae (SL) were deposited closer to root tips of NHE compared with HE. Interestingly, the vessel-to-CS overlap was identified as an ABA-driven anatomical trait modified in response to actual conditions. Results of correlation analyses and exogenous applications of ABA/Abamine indicate that ABA regulates development of both types of apoplastic barriers through promoting activities of phenylalanine ammonialyase, peroxidase and expressions of suberin-related genes (SaCYP86A1, SaGPAT5, SaKCS20). Using SIET and PTS tracer confirmed that ABA-promoted deposition of CSs and SL significantly reduced Cd entrance into root stele. Therefore, maintenance of low ABA levels in HE suppressed development of apoplastic barrier and maximized Cd uptake via apoplastic pathway.