Calcium homeostasis is disturbed emanating from altered calciotropic hormone concentrations in type 2 diabetes mellitus (T2DM), a condition preceded by pre-diabetes. Disrupted calcium homeostasis has shown to promote insulin resistance and hyperglycaemia. However, the changes to calciotropic hormones in the pre-diabetic state is not known. Hence, this study investigated the effects of diet- induced pre-diabetes on calciotropic hormones in a pre-diabetic rat model. This study also sought to determine the association of calciotropic hormones with glycated haemoglobin (HbA1c) and insulin resistance during pre-diabetes. Twelve male Sprague Dawley rats were randomly assigned into two groups (n=6, per group): pre-diabetic (PD) and non-pre-diabetic (NPD). Fasting blood glucose (FBG), insulin, HbA1c and the homeostatic model assessment for insulin resistance (HOMA-IR) in addition to urine calcium, plasma calcium, parathyroid hormone (PTH), calcitonin, vitamin D and calcitriol concentrations were analysed at week 20. Correlation analysis was performed to examine the associations of calciotropic hormones with HOMA-IR and HbA1c. The results demonstrated increased concentrations of HbA1c, FBG, insulin and HOMA-IR in the PD group by comparison to NPD. Furthermore, plasma PTH, calcitonin, urine calcium, calcitriol and vitamin D levels increased along with unchanged plasma calcium concentrations in the PD group by comparison to NPD. Plasma PTH and calcitonin levels were positively correlated with HbA1c but not with HOMA-IR in the PD group. Plasma calcitriol levels were negatively correlated with HbA1c in the PD group. These observations suggest that calcium homeostasis is disturbed in diet- induced pre-diabetes but the body compensates for the changes by inducing an increase in calciotropic hormone levels. Furthermore, pre-diabetes may promote the development of hyperglycemia in T2DM through altering calciotropic hormone levels.