The present trial was performed to reveal the regulatory effects of L-theanine on the levels of lipopolysaccharide (LPS) endotoxin within different biofluids, as well as relevant inflammatory responses of dairy cattle under heat stress conditions. Thirty lactating Chinese Holstein dairy cattle (189 ± 47 days in milk, and 2 ± 1 parities) were allocated in a completely randomized design to each of 3 dietary treatments: the control (CON, 0 g/d per cow L-theanine), the low L-theanine dosage treatment (LL, 16 g/d per cow L-theanine), and the high L-theanine dosage treatment (HL, 32 g/d per cow L-theanine). This trial consisted of 38 d (7 d for adaption and 31 d for data and sample collection), and sample collection for rumen liquid, blood plasma or serum, and milk were conducted on the d 27 and 38, respectively. Dairy cattle were constantly exposed to environmental heat stress during this experiment according to the recorded temperature-humidity index (THI). In the LL treatment, LPS concentration in rumen liquid was higher (P < 0.05), whilst LPS densities in plasma and milk were lower (P < 0.05) than those of the CON. Supplementing L-theanine at 2 dosages both significantly lowered (P < 0.05) the level of interleukin (IL)-1β in the serum. Results of the present study suggested that L-theanine could be a promising additive in reducing the detrimental effects of heat stress on dairy cows, and L-theanine supplementation at 16 g/d per cow is preferred because it reduced the LPS translocation into the peripheral blood and LPS accumulation in the milk, as well as mitigated LPS-induced inflammatory reactions in dairy cows during heat stress. Further studies are necessitated to investigate the underlying mechanisms of L-theanine in LPS alteration and inflammation alleviation.