The growing demand for lithium (Li) as a key component in battery production is raising concerns about the possible increase in its level in the environmental matrices. Sustainable processes for the remediation of metal-contaminated sites have been targeted, and plants could be useful for this scope. In this regard, the selection of plant material with an improved ability to tolerate and accumulate Li is required. In this work, a proof-of-concept in microshoots cultivated in vitro condition was performed to evaluate the potential of hemp (Cannabis sativa L.) to grow and accumulate Li when exposed to relevant concentrations of this metal in the medium. The results demonstrated the toxic effect of Li on plant growth even at the lowest Li level tested (50 mg L^− 1). Increased Li concentrations (150 and 300 mg L^− 1) caused an impairment of the photosynthetic machinery and an alteration of element uptake, particularly for micronutrients such as Cu, Fe, Zn, Ni, and Co. A remarkable accumulation of Li in the microshoots was detected in relation to the metal concentration in the substrate. The overall data were discussed, providing evidence for the potential of hemp plants to tolerate and accumulate Li. The results of this work could be a reference for further insights into the potential of hemp for Li phytoremediation and phytomining, although the in vitro growth conditions allow them to be highlighted as preliminary indications.