The vertebrate pineal gland is dedicated to the production of the hormone melatonin, which increases at night to influence circadian and seasonal rhythms. This increase is associated with dramatic changes in the pineal transcriptome. Here, single-cell analysis of the rat pineal transcriptome was approached by sequencing mRNA from ~17,000 individual pineal cells, with the goals of profiling the cells that comprise the pineal gland and examining the proposal that there are two distinct populations of pinealocytes differentiated by the expression of Asmt, which encodes the enzyme that converts N-acetylserotonin to melatonin. In addition, this analysis provides evidence of cell-specific time-of-day dependent changes in gene expression. Nine transcriptomically distinct cell types were identified: ~90% were classified as melatonin-producing α- and β-pinealocytes (1:19 ratio). Non-pinealocytes included three astrocyte subtypes, two microglia subtypes, vascular and leptomeningeal cells, and endothelial cells. α-Pinealocytes were distinguished from β-pinealocytes by ~3-fold higher levels of Asmt transcripts. In addition, α-pinealocytes have transcriptomic differences that likely enhance melatonin formation by increasing the availability of the Asmt cofactor S-adenosylmethionine, resulting from increased production of a precursor of S-adenosylmethionine, ATP. These transcriptomic differences include ~2-fold higher levels of the ATP-generating oxidative phosphorylation transcriptome and ~8-fold lower levels of the ribosome transcriptome, which is expected to reduce the consumption of ATP by protein synthesis. These findings suggest that α-pinealocytes have a specialized role in the pineal gland: efficiently O-methylating the N-acetylserotonin produced and released by β-pinealocytes, thereby improving the overall efficiency of melatonin synthesis. We have also identified transcriptomic changes that occur between night and day in seven cell types, the majority of which occur in β-pinealocytes and to a lesser degree in α-pinealocytes; many of these changes were mimicked by adrenergic stimulation with isoproterenol. The cellular heterogeneity of the pineal gland as revealed by this study provides a new framework for understanding pineal cell biology at single-cell resolution.