Abstract Cilia are complex microtubule-based organelles implicated in the aetiology of numerous diseases. Accordingly, many cilia-associated proteins have been described, while those distinguishing cilia subtypes are poorly defined. Here, we characterise the gene signature associated with human motile cilia that captures both known and unknown components of this class of cilia. To define the signature, we performed network deconvolution of transcriptomics data derived from tissues possessing motile ciliated cell populations. For each tissue, genes coexpressed with the motile cilia-associated transcriptional factor, FOXJ1 , were identified. The consensus across tissues provided a transcriptional signature of 248 genes. For validation, we examined the literature, databases, single cell RNA-Seq data, and the localisation of mRNA and proteins in motile ciliated cells. To validate some of the many poorly characterised genes, we performed new localisation experiments on ARMC3 , EFCAB6 , FAM183A , MYCBPAP, RIBC2 and VWA3A . In summary, we report a highly validated set of motile cilia-associated genes that helps shape our understanding of these complex cellular organelles. Summary This work defines a conserved transcriptional signature associated with human motile cilia, including many genes with little or no previous association with these structures. These genes were compared with existing resources and a number of poorly characterised genes validated. Graphical abstract