The objective was to investigate the effects of rumen-protected Met (RPM) during a heat stress (HS) challenge on the response of mammary gland explants to lipopolysaccharide (LPS). Thirty-two multiparous, lactating Holstein cows (184 ± 59 DIM) were randomly assigned to 1 of 2 environmental treatment groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc., France; 0.105% DM of TMR as top dress) or TMR without RPM] in a crossover design. The study was divided into 2 periods with 2 phases per period. During phase 1 (9d), all cows were in thermoneutral conditions (TN; THI = 60 ± 3) and fed ad libitum. During phase 2 (9d), group 1 (n = 16) was exposed to HS using electric heat blankets. Group 2 (n = 16) remained in TN (THI = 61 ± 4) but was pair-fed to HS counterparts. After a 14d washout and 7d adaptation period, the study was repeated (period 2). Environmental treatments were inverted relative to phase 2 in period 1 (sequence), while the dietary treatments remained the same. Mammary tissue was harvested at the end of phase 2. Twenty-five mg of tissue per cow was incubated with 0 or 3 μg/mL of LPS for 2h. Statistical analysis was performed using the MIXED procedure of SAS (Cary, NC.). Regardless of Met supplementation, incubation with LPS increased mRNA abundance of interleukin-8, 6, 1β, C-X-C motif chemokine ligand 2 (CXCL2), tumor necrosis factor α, nuclear factor kappa B subunit 1 (NFKB1) and toll-like receptor 2 (P < 0.001). An environment × LPS interaction was observed for NFKB1 (P = 0.03); expression was greater in LPS-treated explants from non-HS compared with HS cows. mRNA abundance of CXCL2, NFKB1, inducible nitric oxide, nitric oxide synthase 2, and superoxide dismutase 2 (P < 0.05) decreased with HS. While LPS did not alter abundance of genes in NFE2L2 signaling (P > 0.10), explants from HS cows had lower abundance of NFE2L2 (P < 0.001) and cullin 3 (P = 0.04) (an inhibitor of NFE2L2). Overall, preliminary evaluation indicates that HS reduced immune and antioxidant responses while RPM did not attenuate the inflammatory response induced by LPS in vitro.