The excessive amount of zeros in single-cell RNA-seq (scRNA-seq) data includes real zeros due to the on-off nature of gene transcription in single cells and dropout zeros due to technical reasons. Existing differential expression (DE) analysis methods cannot distinguish these two types of zeros. We developed an R package DEsingle which employed Zero-Inflated Negative Binomial model to estimate the proportion of real and dropout zeros and to define and detect three types of DE genes in scRNA-seq data with higher accuracy.