Vitamin D3is a fat-soluble prohormone that is activated inside the liver to produce 25-hydroxyvitamin D3(calcidiol), and in the kidney to produce the fully active 1α, 25-dihydroxy vitamin D3(calcitriol). A previous work piloted in our laboratory, resulted in a successful recovery of a local soil-promisingActinomyces hyovaginalisisolate CCASU-A11-2 capable of converting vitamin D3into calcitriol. Despite the rising amount of research on vitamin D3bioconversion into calcitriol, further deliberate studies on this topic can significantly contribute to the improvement of such a bioconversion process. Therefore, this work aimed to improve the bioconversion process, using the study isolate, in a 14 L laboratory fermenter (4 L fermentation medium composed of fructose (15 g/L), defatted soybean (15 g/L), NaCl (5 g/L), CaCO32 g/L); K2HPO4, (1 g/L) NaF (0.5 g/L) and initial of pH 7.8) where different experiments were undertaken to investigate the effect of different culture conditions on the bioconversion process. Using the 14 L laboratory fermenter, the calcitriol production was increased by about 2.5-fold (32.8 µg/100 mL) to that obtained in the shake flask (12.4 µg/100 mL). The optimal bioconversion conditions were inoculum size of 2% v/v, agitation rate of 200 rpm, aeration rate of 1 vvm, initial pH of 7.8 (uncontrolled); addition of vitamin D3(substrate) 48 h after the start of the main culture. In conclusion, the bioconversion of vitamin D3into calcitriol in a laboratory fermenter showed a 2.5-fold increase as compared to the shake flask level where, the important factors influencing the bioconversion process were the aeration rate, inoculum size, the timing of substrate addition, and the fixed pH of the fermentation medium. So, those factors should be critically considered for the scaling-up of the biotransformation process.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13568-023-01574-3.