Connectomic analysis of the nervous system aims to discover and establish principles that underpin normal and abnormal neural connectivity and function. In Hirst et al (2013) we performed image analysis of motor unit connectivity in the fourth deep lumbrical muscle (4DL) of mice, using transgenic expression of fluorescent protein in motor neurones as a morphological reporter. We developed a method that accelerated segmentation of confocal image projections of 4DL motor units, by applying high resolution (63×, 1.4 NA objective) imaging or deconvolution only where either proved necessary, in order to resolve axon crossings that produced ambiguities in the correct assignment of axon terminals to identified motor units imaged at lower optical resolution (40×, 1.3 NA). This dataset contains microscopy images which show Yellow Fluorescent Protein 16 (YFP-16) expression in muscle 21.9-2L of the mouse footpad. The remainder of the data relating to this study can be found grouped together in the "Connectomic analysis of the mouse fourth deep lumbrical muscle connectome" DataShare Collection at http://datashare.is.ed.ac.uk/handle/10283/2657.