Biological sources of pigments receive major attention nowadays because of the stringent rules and regulations applied to chemically synthesized pigments. The aims of this study were isolating carotenoids producing Micrococcus spp. and Rhodotorula spp. from soil sources, optimizing the culture conditions for biomass and carotenoids production, molecular cloning of Crt gene, characterizing yielded pigment, and investigating their anti-cancer activities on human breast cancer cells. Carotenoid producing strains, M. luteus and R. mucilaginosa, were isolated from the soil and sediment samples in Kerman Province, Iran; they were identified using 16srDNA analysis. Optimum conditions for biomass and carotenoids production were determined. Further, Crt gene cloned into pTG19 vector and the effect of carotenoid pigments on MCF-7 breast cancer cell line were evaluated. The optimum growth and carotenoids production by strains were 25°C and pH 7.0 for M. luteus, and 25°C and pH 6.5 for R. mucilaginosa. FTIR and TLC analysis showed high similarity of extracted pigments with carotenoids. The expression levels of Crt genes mRNA found to be approximately 5-fold higher in transformed E. coli in comparison with M. luteus and R. mucilaginosa (p <0.001). MCF-7 cells viability decreased in a concentration and time dependent manner (p <0.05). The doses IC50 against MCF-7 cells for yellow and pink pigments were calculated to be 1426.69 µg/ml (r2=0.95) and 1412.1 µg/ml (r2=0.92), respectively. Microorganisms presented in this study can be used as potential sources of commercial carotenoids production and antitumor metabolites.