Simple SummaryCyclosporaspp. is an important zoonotic parasite that poses a threat to public health. At present, the research onCyclosporaspp. in cattle of the Yunnan Province remains limited. We firstly reported the prevalence ofCyclosporaspp. in Holstein cattle in the Yunnan Province, China. In order to understand the prevalence and genotype ofCyclosporaspp. in the Yunnan Province, 524 fecal samples from 4 regions were collected and tested. The results of the present study showed that 13 samples were positive forCyclosporaspp., and the total infection rate ofCyclosporaspp. was 2.48%. We analyzed different risk factors, such as regions, sexes, and ages, but the difference was not statistically significant. Phylogenetic analysis showed that fiveCyclosporaspp. samples were classified into the zoonotic group ofCyclospora cayetanensis, which had zoonotic potential. The results of the present study indicated thatCyclosporaspp. in the Holstein cattle of Yunnan Province posed a risk of zoonosis.AbstractCyclosporaspp. is a food-borne intestinal protozoan, which is widely distributed in the world and poses the risk of zoonosis. In order to reveal the prevalence ofCyclosporaspp. in Holstein cattle in partial areas of the Yunnan Province, 524 fresh fecal samples of Holstein cattle were collected from Dali, Kunming, Chuxiong, and Qujing in Yunnan Province. A nested PCR amplification of the small subunit (SSU) rRNA gene ofCyclosporaspp. was carried out, and the products of the nested PCR were further analyzed by restriction fragment length polymorphism (RFLP) usingBsp EⅠ. The results of the present study showed that 13 samples were positive forCyclosporaspp., and the total infection rate ofCyclosporasp. was 2.48%. The infection ofCyclosporaspp. was detected in Dali, Qujing, and Chuxiong. Chuxiong showed the highest infection rate (5.71%), and infection rate in Dali and Qujing was 2.19% and 3.16%, respectively. Interestingly, the infection ofCyclosporaspp. was not detected in Kunming. The infection ofCyclosporaspp. showed no significant differences among different regions (p> 0.05).Cyclosporasp. infection was detected in all ages and sexes, but the differences were not significant (p> 0.05). Sequencing and phylogenetic analysis showed that fiveCyclosporaspp. samples were closely related to theCyclosporaspp. of humans, and the others were closely related to theCyclosporaspp. of bovines. The results of the present study suggested that there was an infection ofCyclosporaspp. in Holstein cattle in the Yunnan Province, and theCyclosporaspp. showed a risk of zoonosis. Thus, the prevention and control ofCyclosporaspp. should be strengthened in the Yunnan Province, China. The results of this investigation provide data references for the further research of Cyclosporiasis in Holstein cattle in the Yunnan Province.