Background and aims: To study the effect of serum thyrotropin (Sr.TSH) and serum cholesterol on gallbladder contraction by cholescintigraphy. Methods: This is a prospective cross-sectional study comprising 77 patients for evaluation of gallbladder function in patients with biliary pain. All of them underwent 99mTc- Mebrofenin fatty meal cholescintigraphy following a standard institutional protocol apart from regular investigations. Dynamic images were acquired for 90 minutes with a standardised fatty meal given at the 60th minute. Gallbladder emptying kinetics was determined with Gallbladder Ejection Fraction (GBEF) calculated at 30 minutes post-meal. Relevant statistical methods were applied for interpretation. Results: The mean of GBEF was 60.82% ± 16.65% and the mean of Sr.TSH was 3.72 ± 3.88 mIU/ml, there was a negative correlation between GBEF and Sr.TSH (Pearson correlation coefficient r = -0.535, p < 0.05). The mean serum cholesterol was 180.61 ± 46.34 mg/dl, there was a negative correlation between GBEF and serum cholesterol (Pearson’s correlation coefficient r = -0.685, p < 0.05). The mean GBEF in euthyroid and hypothyroid patients were 65.04% ± 15.04% and 47.27% ± 15.93% respectively (P < 0.05). Conclusion: Hypothyroidism is one of the key factors affecting gallbladder contractility and should not be overlooked in the management of functional gallbladder disorders. Cholescintigraphy can accurately assess gallbladder function qualitatively and quantitatively, and it can help (aid) physicians in diagnosing and decision-making on the management of biliary pain in hypothyroidism.