Our objective was to compare mammary tissue gene expression profiles during a Streptococcus uberis (S. uberis) mastitis challenge between lactating cows subjected to dietary-induced negative energy balance (NEB; n = 5) and cows fed ad libitum to maintain positive energy balance (PEB; n = 5) to better understand the mechanisms associated with NEB and risk of mastitis during the transition period. The NEB cows were feed-restricted to 60% of calculated net energy for lactation requirements for 7 days, and cows assigned to PEB were fed the same diet for ad libitum intake. Five days after feed restriction, one rear mammary quarter of each cow was inoculated with 5,000 cfu of S. uberis (O140J). At 20 h postinoculation, S. uberis-infected mammary quarters from all cows were biopsied for RNA extraction. Negative energy balance resulted in 287 differentially expressed genes (DEG; false discovery rate ≤ 0.05), with 86 DEG upregulated and 201 DEG downregulated in NEB vs. PEB. Canonical pathways most affected by NEB were IL-8 signaling (10 genes), glucocorticoid receptor signaling (13), and NRF2-mediated oxidative stress response (10). Among the genes differentially expressed by NEB, cell growth and proliferation (48) and cellular development (36) were the most enriched functions. Regarding immune response, HLA-A was upregulated due to NEB, whereas the majority of genes involved in immune response were downregulated (e.g., AKT1, IRAK1, MAPK9, and TRAF6). This study provided new avenues for investigation into the mechanisms relating NEB and susceptibility to mastitis in lactating dairy cows.