Arsenic (As) pollution seriously threatens human and ecological health. Microalgal cell wall and extracellular polymeric substances (EPS) are known to interact with As, but their roles in the As resistance, accumulation and speciation in microalgae remain unclear. Here, we used two strains of Chlamydomonas reinhardtii, namely CC-125 (wild type) and CC-503 (cell wall-deficient mutant), to examine the algal growth, EPS synthesis, As adsorption, absorption and transformation under 10–1000 µg/L As(III) and As(V) treatments for 96 h. In both strains, the As absorption increased after the EPS removal, but the growth, As adsorption, and transformation of C. reinhardtii declined. The CC-125 strain was more tolerant to As stress and more efficient in EPS production, As accumulation, and redox transformation than CC-503, irrespective of EPS presence or absence. Three-dimension excitation-emission matrix (3D-EEM) and attenuated total reflectance infrared spectroscopy (ATR-IR) analyses showed that As was bound with functional groups in the EPS and cell wall, such as -COOH, NH and -OH in proteins, polysaccharides and amino acids. Together, this study demonstrated that EPS and cell wall acted as barriers to lower the As uptake by C. reinhardtii. However, the cell wall mutant strain was more susceptible to As toxicity due to lower EPS induction and higher As absorption.