Heat stress (HS) has been shown to reduce immune functions, however, feeding rumen-protected methionine (RPM), a known immunostimulant, may mitigate its effects. Thirty-two multiparous, lactating Holstein cows (DIM 184 ± 59) were randomly assigned to 1 of 2 environmental groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc.); 0.105% DM of TMR as top dress) or without (CON)] in a crossover design, with 2 periods and 2 phases per period. In phase 1 (9d), cows were in thermoneutral conditions (TN; 16.0 ± 2.5°C, 71.4 ± 7.5% humidity, THI = 60 ± 3) and fed ad libitum. In phase 2 (9d, 16.8 ± 2.5°C, 66.8 ± 8.0% humidity, THI = 61 ± 4), group 1 (n = 16) was exposed to HS using electric heat blankets (THI = 89 ± 3). Group 2 (n = 16) remained in TN but was pair-fed to HS counterparts. After a 21d washout period, the study was repeated (period 2). Environmental, but not dietary treatments were inverted (sequence effect). Blood was sampled 6d into period 2 and incubated with PI-labeled E. coli. Cells were stained to segregate neutrophils (CH138A) and monocytes (CD14) via flowcytometry. DHR was used as indicator of oxidative burst. Data were analyzed using PROC MIXED in SAS, with diet, environment, and their interaction as main effect, as well as period and sequence to account for the crossover design. Cow was used as random effect. HS increased (P < 0.001) rectal temperature and respiration rate (+0.3°C, +13.7 breaths/min) compared with CON. RPM did not affect (P > 0.05) functionality of both cell type. HS, instead, decreased neutrophil (P = 0.01, 72 vs 64%) and monocyte (P = 0.001, 27 vs 19%) oxidative burst and monocyte phagocytosis capacity (P = 0.05, 10 vs 8%), while it only tended to decrease (P = 0.11, 9 vs 7%) neutrophil phagocytosis. A sequence effect was detected for both monocyte (P = 0.01, 19 vs 27%) and neutrophil (P = 0.03, 64 vs 73%) oxidative burst, with overall lower response in animals subjected to HS in period 1. Results highlight the negative effect of HS on dairy cow innate immune function, including a probable carry over chronic effect. RPM was ineffective in counteracting the detrimental effect of HS during late-lactation, probably due to the short feeding period before the imposed stress.