AbstractThe insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1-R) play key roles in growth, regulation of nutrient metabolism and carbohydrate homeostasis. Insulin-like molecules in prokaryotes and other early life have been reported. However, an account of metabolic effects of insulin, transcriptomic evidence of expression of glucose transporting channels (GLUT) and homology modelling of IR and IGF1-R like proteins in unicellular life-forms have yet to be established. Acanthamoeba spp. has existed for about 2 billion years and is one of the earliest mitochondriate unicellular eukaryotic cells on Earth. Despite Acanthamoeba spp. being grown in a medium called peptone-yeast-glucose (PYG) for over 50 years, the mechanism and regulation of glucose uptake by IR or IGF1-R molecules in this microbe has not yet been reported. Several methods were utilized to validate the effects of insulin on trophozoites of A. castellanii, including: growth assays with insulin, estimation of glucose and potassium (K+) entry into the cell, and histology showing anabolic effects on proteins. Bioinformatic computational tools and homology modeling demonstrated the involvement of IR like proteins, GLUT, and adapter proteins in mediating the IR cascade. Growth assays showed proliferative effects in a dose range of 2.98–5.97 µmol/mL of insulin. After insulin exposure, A. castellanii trophozoites displayed enhanced Periodic acid-Sciff (PAS) staining. Amino acid sequence similarities and homology modelling revealed ACA1_163470 in Acanthamoeba spp. to be a homolog of human-IR. Acanthamoeba protein ACA1_336150 shares similarities with IGF1-R. Additionally, some proteins like ACA1_060920 have attributes of GLUT like channels on homology modelling and show similarity with human GLUT. Knowledge of IR and insulin effects in Acanthamoeba spp. contributes to its biology and advances current understanding behind the evolution of IR and IGF1-R signalling cascade.