The vertebrate pineal gland produces the hormone melatonin, which increases at night to influence circadian rhythms. This is associated with dramatic changes in the pineal transcriptome which have not yet been examined at the single-cell level. Here, analysis of the rat pineal gland was approached by sequencing mRNA from thousands of individual pineal cells. Nine transcriptomically distinct cell types were identified: ~90% were classified as melatonin-producing α- and β-pinealocytes (1:17). The remaining non-pinealocytes included three astrocyte subtypes, two microglia subtypes, vascular leptomeningeal cells, and endothelial cells. α- and β-Pinealocytes exhibited differences in several functional areas. α-pinealocytes had features that are likely to elevate ATP, including ~3-fold higher levels of the ATP-generating oxidation phosphorylation transcriptome, ~7-fold lower levels of 60 transcripts in the ATP-consuming ribosome transcriptome, reduced levels of G-protein γ-subunit transcripts and reduced night/day differential expression. In addition, α-pinealocytes had ~3-fold higher levels of Asmt, the enzyme that converts N-acetylserotonin to melatonin. These ATP-increasing modifications are likely to elevate the catalytic potential of Asmt, as ATP is a precursor of the Asmt cofactor S-adenosyl methionine (SAM). These findings suggest that α-pinealocytes are specialized to efficiently O-methylate N-acetylserotonin produced by both pinealocyte subtypes, thereby improving the efficiency of melatonin synthesis. We have also identified transcriptomic changes occurring between night and day in seven cell types, most of which occur in α-pinealocytes and, to a greater degree, β-pinealocytes. These results provide novel insights into the cellular heterogeneity of the pineal gland and provide a framework for understanding pineal cell biology at single-cell resolution.