Iron (Fe) is an essential micronutrient for plant survival and proliferation. Plants have evolved complex mechanisms to maintain Fe homeostasis in response to Fe deficiency. In this study, we evaluated the physiological, biochemical and transcriptomic differences between poplars grown under Fe-sufficient and Fe-deficient conditions to elucidate the mechanistic responses of poplars to Fe deficiency. Our results revealed that chlorophyll synthesis and photosynthesis were inhibited under Fe-deficient conditions. The inhibition of these pathways caused chlorosis and reduced shoot growth. Although both photosynthetic systems (PSI and PSII) were inhibited under Fe limitation, PSI was affected more severely and earlier than PSII. Fe deficiency also promoted root growth and increased the accumulation of divalent metal ions in roots. IRT1 and NRAMP1 are both Fe²⁺ transporters for iron uptake in Arabidopsis. In this study, however, only NRAMP1was induced to promote Fe²⁺ uptake in roots at the late stage of Fe deficiency response. It indicated that NRAMP1, rather than the more well-known IRT1, might be a major Fe²⁺ transporter at the late stage of Fe-deficiency in poplars.