Growth chamber experiments were conducted to evaluate the effects of relative humidity and temperature on the efficacy, absorption, and translocation of glufosinate at 205, 410, and 820 g ha−1 in Palmer amaranth, redroot pigweed, and common waterhemp. Low relative humidity decreased control of all three species by glufosinate. However, control increased as application rate increased at low relative humidity. Amaranth species grown under 21/16, 26/21, and 31/26 C day/night temperature regimes responded differently to glufosinate. At 26/21 C, glufosinate at 820 g ha−1 controlled redroot pigweed less effectively than it controlled Palmer amaranth and common waterhemp, whereas at 410 g ha−1, glufosinate controlled common waterhemp more effectively than it controlled the other two species. Neither temperature nor relative humidity altered the absorption of 14C-glufosinate in any of the three species. Most of the absorbed glufosinate remained in the treated leaves at all three temperature regimes and two relative humidity levels. However, glufosinate translocation was greater in plants grown at 90% than in those grown at 35% relative humidity, and this phenomenon coincided with greater control of the amaranth species at the high humidity level. The study showed that relative humidity had a greater effect than temperature on glufosinate toxicity to Palmer amaranth, redroot pigweed, and common waterhemp.