In recent decades, fertility in dairy cows has declined as cows have been highly selected for milk production. Dairy cows often experience a state of negative energy balance (NEB) during the first few months of lactation resulting in dramatic fluctuations in circulating levels of a variety of hormones and metabolites. The systemic hormonal and metabolic dysregulation can also have a profound effect on the ovarian follicular micro-environment which can affect the molecular composition and developmental capacity of the oocyte, which is highly responsive to its micro-environment during growth and maturation. The aim of this study was to identify how the stage of lactation and energy status affect gene expression in individual in vivo-matured oocytes. Oocytes were collected by ovum pick-up (OPU) once from each of 8 Holstein heifers at the age of 14 mo, and 3 times from 13 lactating Holstein cows at 47, 75, and 130 d in milk. RNA was extracted from each single oocyte with a total of 19 oocytes from heifer and 119 oocytes from cows for relative transcript abundances for each of 64 genes from a variety of functional categories such as metabolism, apoptosis, redox, heat shock, and epigenetics. Gene expression was evaluated by reverse transcription and quantitative real-time PCR using the powerful BioMark platform for single-cell gene expression analysis from Fluidigm (South San Francisco, CA). Raw qPCR data were normalized and analyzed using the comparative CT method. Out of the 64 genes tested, GLRX, H19, HSPA1A, and NDN were found to be different (adj. P < 0.05) between cows and heifers and within lactating cows; ACACA, CAT, EZH2, G6PD, GLRX, GLRX3, and SOD1 were found to be significantly affected by the stage of lactation; and DNMT1, DNMT3B, GLUD1, GSR, HSP90A1, and SUV39H1 were found to be significantly affected by the BHB level within lactating cows. We conclude that lactation and progression in milk production cause a sort of metabolic stress that directly or indirectly alters the molecular constituents of oocytes and thereby potentially impact oocyte developmental competence.