Neospora caninum is an important pathogen commonly causing spontaneous abortion in livestock. The parasite is known to remain in cysts in an inactive state; or it can undergo expansive development within an intermediate host. However, the mechanisms that trigger the proliferation of N. caninum have not been thoroughly elucidated. For various organisms, it has been demonstrated that microRNAs (miRNAs) can act as important endogenous regulatory factors in gene regulation during cell differentiation and development. However, miRNAs and their function have not been studied in N. caninum. In this study, small RNA libraries from N. caninum tachyzoites (NC-1 strain) were analyzed using a high-throughput RNA sequencing technology combined with systematic bioinformatics analysis. A considerable number of novel miRNAs from N. caninum NC-1 strain tachyzoites were identified. Of the 300 miRNAs found by bioinformatics analysis, 10 were conserved miRNAs belonging to 10 metazoan miRNA families, while 290 were novel miRNAs. The expression of 13 novel miRNAs was verified by real-time quantitative PCR (qRT-PCR). Data from this study provided and identified authentic miRNAs for the first time in N. caninum. The study also introduces a framework for further investigations of RNAi-dependent regulatory mechanisms of the parasite and provides data for further understanding of N. caninum development.