Phytochrome photoreceptors mediate adaptive responses of plants to red and far-red light. These responses generally entail light-regulated association between phytochromes and other proteins, among them the phytochrome-interacting factors (PIF). The interaction with phytochrome B (PhyB) localizes to the bipartite APB motif of the PIFs (PIF). To address a dearth of quantitative interaction data, we construct and analyze numerous PIF3/6 variants. Red-light-activated binding is predominantly mediated by the APB N-terminus, whereas the C-terminus modulates binding and underlies the differential affinity of PIF3 and PIF6. We identify PIF variants of reduced size, monomeric or homodimeric state, and with PhyB affinities between 10 and 700 nM. Optogenetically deployed in mammalian cells, the PIF variants drive light-regulated gene expression and membrane recruitment, in certain cases reducing basal activity and enhancing regulatory response. Moreover, our results provide hitherto unavailable quantitative insight into the PhyB:PIF interaction underpinning vital light-dependent responses in plants.