Sickle cell disease (SCD) is one of the most common autosomal-recessive genetic disorders affecting millions worldwide. The three most significant abnormal features of RBCs from SCA patients are haemoglobin (HbS) polymerisation, phosphatidylserine (PS) exposure and significantly increased cation permeability via the KCl cotransporter (KCC), and are critical to the pathogenesis of SCD. The outcome of these abnormal features usually results in altered red blood cell (RBC) fragility and rheology, increased viscosity with blockage of small capillary vessels, and changed RBC membrane permeability. This study aimed to explore novel aspects of these three features with respect to their modulation by physiological stimuli experienced in vivo and a major pharmacological modulator, protein phosphorylation. Various physiological stimuli such as oxygen tension (PO2), rate of deoxygenation, pH, urea, anisotonicity and medium composition, play an important role in regulation of HbS polymerisation, PS exposure and KCC activity in HbSS RBCs. In addition, low potassium (LK) sheep RBCs were utilised as an animal model to examine these physiological stimuli on KCC activity in comparison with their effects on human HbSS RBCs’. Various pharmacological manoeuvres were also tested for their effects on HbS polymerisation, PS exposure and KCC activity. A key finding was the marked stimulatory effect of the pan “with no lysine” kinase inhibitor WNK463 on KCC activity in HbSS RBCs, indicative of a critical role for WNK kinases in control of KCC in RBCs. However, there was no pharmacological evidence for the downstream involvement of SPAK/OSR1, although definitive evidence awaits phosphoproteomic studies. Overall, results provide a better and more comprehensive understanding of the pathogenesis of SCD with the potential to inform novel therapeutic approaches.