Vitamin D3 is a crucial co-regulator of bone growth and remodeling, neuromuscular function, inflammation, proliferation, differentiation and apoptosis of cells. Intensive research on endogenous sulfur metabolism has revealed that hydrogen sulfide (H2S) is an important modulator of various physiological processes in mammals. Noteworthy, these compounds are perceived as potential agents in the treatment of numerous disorders, including cardiovascular diseases and different types of cancer. The interaction between vitamin D3 and H2S is unknown. The aim of the study is to assess the influence of cholecalciferol (vitamin D3, calcitriol) on H2S tissue concentrations in mouse brain, heart and kidney. Twenty four SJL mice were given intraperitoneal injections of cholecalciferol at 10000 IU/kg body weight (b.w.) per day (group A, n = 8) or 40000 IU/kg b.w. per day (group B, n = 8). The control group (n = 8) received physiological saline. Free H2S tissue concentrations were measured via the SIEGEL spectrophotometric modified method. There was a significant progressive increase in the H2S concentration along with the rising cholecalciferol doses as compared to the control group in the heart (by 29.6% and by 74.1%, respectively). Higher vitamin D3 dose caused H2S accumulation in the brain (by 10.9%) and in the kidney (by 10.1%). Our study has proven that cholecalciferol affects H2S tissue concentration in different mouse organs.