Single-cell mass cytometry significantly increases the dimensionality of cytometry analysis as compared to fluorescence flow cytometry, providing unprecedented resolution of cellular diversity in tissues. However, analysis and interpretation of these high-dimensional data poses a significant technical challenge. Here, we present cytofkit, a new Bioconductor package, which integrates both state-of-the-art bioinformatics methods and in-house novel algorithms to offer a comprehensive toolset for mass cytometry data analysis. Cytofkit provides functions for data pre-processing, data visualization through linear or non-linear dimensionality reduction, automatic identification of cell subsets, and inference of the relatedness between cell subsets. This pipeline also provides a graphical user interface (GUI) for ease of use, as well as a shiny application (APP) for interactive visualization of cell subpopulations and progression profiles of key markers. Applied to a CD14-CD19- PBMCs dataset, cytofkit accurately identified different subsets of lymphocytes; applied to a human CD4+ T cell dataset, cytofkit uncovered multiple subtypes of TFH cells spanning blood and tonsils. Cytofkit is implemented in R, licensed under the Artistic license 2.0, and freely available from the Bioconductor website, https://bioconductor.org/packages/cytofkit/. Cytofkit is also applicable for flow cytometry data analysis.