Abstract Ultraviolet-B (UV-B) radiation negatively affects photosynthesis; however, some studies have also demonstrated positive effects of UV-B radiation on photosynthesis. In cyanobacteria, histidine kinase Hik33 plays an important role in mediating responses to multiple environmental stresses, but whether Hik33 is involved in the positive regulation of photosynthesis by UV-B remains unclear. Here, we successfully established CRISPR-Cpf1-based genetic transformation in the UV-B-tolerant cyanobacterium Nostoc sphaeroides CCNUC1, paving the way for unveiling the regulatory mechanism of UV-B-induced photoacclimation. We constructed a nshik33 knockout mutant that lacked the UV-B-induced positive effects on growth, photosynthetic activity, and cyclic electron flow around photosystem I seen in non-acclimated control cells. We determined that nsHik33 attenuates the phosphorylation of nsRpaB under low UV-B light, thereby upregulating the transcription of photosynthesis-associated genes (such as psbA and psaA, encoding core subunits of photosystems II and I, respectively) in Nostoc sphaeroides CCNUC1. To investigate the conservation of nsHik33 function, we heterologously expressed nshik33 of N. sphaeroides CCNUC1 in the Δhik33 mutant of Synechocystis sp. PCC 6803 and discovered that nsHik33 complements the defective photosynthesis phenotype of Δhik33 mutant cells acclimated to low UV-B. These data indicate that Hik33 plays a crucial role in UV-B-induced photosynthetic acclimation in cyanobacteria.