The purpose of this study is to determine neural, vascular, protein secretion, and cellular signaling changes with disease progression in lacrimal glands of the thrombospondin-1-/- (TSP-1-/-) mouse model of dry eye compared to C57BL/6 wild-type (WT) mice. Neural innervation was reduced in TSP-1-/- lacrimal glands compared to WT controls, whereas the number of blood vessels was increased. Intracellular Ca2+ stores and the amount of lysosomes, mitochondria, and secretory granules, but not the endoplasmic reticulum, were reduced in TSP-1-/- compared to WT acini at 12 weeks of age. Ex vivo high KCl-evoked secretion was decreased in TSP-1-/- compared to WT lacrimal gland tissue pieces. The α1D-adrenergic agonist-stimulated response was increased in TSP-1-/- at 4 and 24 weeks but decreased at 12 weeks, and the ATP and MeSATP-stimulated peak [Ca2+]i responses were decreased at 24 weeks. These changes were observed prior to the appearance of mononuclear infiltrates. We conclude that in the lacrimal gland the absence of TSP-1: injures peripheral nerves; blocks efferent nerve activation; decreases protein secretion; and alters intracellular Ca2+ stores. Through these effects the absence of TSP-1 leads to disruption of ocular surface homeostasis and development of dry eye.