Light, as the energy and signal sources for plant growth and development, is one of the most important environment factors in recently developed plant factories with artificial light (PFALs). To find the optimal combination of light wavelengths for lettuce (Lactuca sativa cv. ‘Tiberius’) plant growth in a PFAL, four treatments, each using red (R; 662 nm) and blue light (B; 447 nm) with a ratio of 4:1 and photon flux density (PFD) of 150 μmol·m−2·s−1, and mixing, respectively, with 50 μmol·m−2·s−1 of green light (G; 525 nm; RBG), yellow light (Y; 592 nm; RBY), orange light (O; 605 nm; RBO) and far-red light (FR; 742 nm; RBFR), were set up during this experiment. A combination of R and B with a ratio of 4:1 and PFD of 200 μmol·m−2·s−1 was set as the control (RB). The responses of lettuce growth, morphology, anatomical structure of the lettuce leaf, photosynthetic performance, lettuce nutritional quality, and energy use efficiency were investigated. The results showed that RBG, RBO, and RBFR increased the shoot fresh weight of lettuce by 20.5%, 19.6%, and 40.4%, and they increased the shoot dry weight of lettuce by 24.2%, 13.4%, and 45.2%, respectively, compared with those under RB. The Pn under RBY was significantly lower than that under RB, although no significant differences in chlorophyll or carotenoid content were found between RBY and RB. RBG increased the lettuce leaf area, the thickness of the leaf palisade tissue, Pn, and light use efficiency compared with those under RB. Plants grown under RBO showed better photosynthetic capacity, such as higher Pn, ΦPSII, and other photosynthetic parameters. RBFR caused an increase in lettuce leaf area and energy use efficiency, but a decrease in leaf thickness and Pn of the single leaf. Moreover, tipburn injury was observed under RBFR. Therefore, these results demonstrate that RBG and RBO can be considered optimal combinations of light wavelengths for lettuce growth in a PFAL in this experiment, although plant growth can also be improved by using RBFR.