Carotenoids are natural pigments synthesized by several microorganisms, including bacteria, yeasts, filamentous fungi and algae. Due to their beneficial biological properties, they are attracting interest from the food, pharmaceutical and cosmetic industries. The biotechnological production of carotenoids includes several advantages, among them, the ability of microorganisms to use low cost substrates as carbon source. Therefore, crude glycerol from biodiesel production has been studied as a carbon source for different microorganisms. In this work, we propose the carotenoids production by fed-batch cultivation using crude glycerol as carbon source. It was used the yeast Rhodotorula mucilaginosa previously isolated by the research group. Two fed-batch strategies were tested: (i) addition of a pulse (10% of initial volume) containing crude glycerol based-medium in the exponential phase of growth (96 h); (ii) addition of a pulse with only crude glycerol in the exponential phase of growth (96 h). Batch cultivation was also performed for comparison. Cultivations were carried out in a rotary shaker (180 rpm, 25°C) using Erlenmeyer flasks (500 mL) with initial volume of 200 mL, evaluating the cell growth, pH and carotenoid production (expressed as μg of β-carotene extracted by L of medium) during the cultivation time (12 days). The pulse containing only crude glycerol did not cause an increase in the carotenoids production, while the pulse of medium provided an increase in the biomass concentration from 8.36 g/L (batch) to 14.77 g/L (fed-batch), resulting in a 4.4% of increase in the carotenoids production from 1152.7 μg/L to 1202.8 μg/L. The maximum specific growth rate increased from 0.047 1/h (batch) to 0.056 1/h (crude glycerol pulse) and 0.058 1/h (medium pulse). That is, fed-batch strategy increased the carotenoid yield and biomass concentration.