Recently, far-red wavelengths (FR, 700–750 nm) have been largely investigated in indoor cultivation systems due to their morphological effects on plants (e.g., leaf expansion and stem elongation), resulting also in increasing yield. This work investigated the effect of substituting part of the red (R) and blue (B) radiation with far-red radiation, while keeping constant the photon flux density, in the light spectrum for lettuce grown in a vertical farm. Lettuce (Lactuca sativa var. Canasta) plants were transplanted and grown in an ebb-and-flow system for 29 days. During the cycle, plants were subjected to five different light treatments: a control treatment consisting of an optimized R and B spectrum (ratio of 3; RB3) with a photosynthetic photon flux density of 200 µmol m−2 s−1, and four treatments in which R and B were partially replaced by 10, 30, 50 and 70 µmol m−2 s−1 of FR light, resulting in an increasing FR fraction. Biomass production and most of the morphological parameters were affected from 15 days after transplanting (DAT), while stomatal conductance from 22 DAT. Leaf greenness and specific leaf area values were influenced by the FR radiation starting from 8 DAT. At 29 DAT, substitution of an amount of R and B photons equal to 30 (RB3–30) or 50 (RB3–50) µmol m−2 s−1 with the same amount of FR radiation resulted in increased leaf biomass in both fresh (+49 and +47%, respectively) and dry weight (+45 and +42%, respectively). With RB3–30, the increase was due to leaf area expansion (+103%), whereas stomatal conductance (gs) and quantum efficiency of photosystem II (ΦPSII) did not change compared with the spectrum with only R and B. With RB3–50, gs and ΦPSII decreased compared with RB3 (-27 and -6%, respectively), but the greater biomass accumulation was supported by the greater leaf expansion (+119 %). The adoption of RB3–30 and RB3–50 also promoted light use efficiency (+45 and +42 %, respectively), lighting energy use efficiency (+48 and +53 %, respectively) and therefore the overall energy performance of the system. The adoption of RB3–30 and RB3–50 is a valid strategy to increase yield for lettuce production, but further studies, also in relation to blue radiation intensity, are needed to avoid the negative effect on leaf pigmentation.