BACKGROUND Jambadyarista is an Ayurvedic polyherbal formulation widely prescribed by Ayurvedic practitioners for the management of diabetes and its associated complications. About 39 companies have marketed this formulation in Bangladesh with consent from the Directorate General of Drug Administration (DGDA). OBJECTIVE This study investigated the sub-acute oral toxicity of Jambadyarista in the Sprague-Dawley rat model. METHODS The sub-acute toxicity studies were executed in Sprague-Dawley rats. Jambadyarista formulation was given for 28-days through oral gavage at 10 mL/kg and 20 mL/kg dose to two different groups comprising 6 rats of both sex/groups. Across the experimental period mortality, adverse reactions were closely monitored. After 28-day feeding hematological, biochemical, and relative organ weights were quantified. RESULTS No mortality and/or signs of morbidity were observed for 28-day of repeated-dose sub-acute toxicity. Any pernicious change in body weight, biochemical, and hematological parameters along with relative organ weight were not observed for Jambadyarista. Correlation study among parameters of the renal profile, liver profile, lipid profile also metabolic hormones (T3 and TSH), and enzymes showed the non-toxic rather beneficial role (hypolipidemic) of Jambadyarista in Sprague-Dawley rats. CONCLUSIONS Jambadyarista preparation did not cause any potential toxic effect in repeated dose subacute toxicity study over Sprague-Dawley rats orally. Therefore, low dose administration of Jambadyarista could have a beneficial effect on diabetes and can be considered safe before the chronic study.