Objectives: At the end of a 60-day course of narrowband UVB phototherapy, administered to individuals with early multiple sclerosis, there were changes in the relative proportions of circulating B-cell subsets. This study investigated phototherapy-associated changes to cytokine responses of B cells when exposed to a TLR7 ligand. Methods: PBMCs from participants of the PhoCIS (Phototherapy for Clinically Isolated Syndrome) trial taken before (day 1) and after phototherapy for 8 weeks (day 60) were incubated with, or without, the TLR7 ligand, R848, for 18 h. Production of TNF and IL-10 in seven B-cell subsets was examined, with cytokine responses in each individual at day 60, adjusted for responses at day 1. Paired PBMCs were from participants administered phototherapy (n = 7) or controls (n = 6). Results: At day 60, significantly fewer B cells, particularly marginal zone-like B cells (CD27+/IgD+), from participants administered phototherapy produced TNF in response to TLR7 stimulation. When responses by seven B-cell subsets were analysed together using multivariate methods, a phototherapy-specific signature was observed. An increased responsiveness from day 1 to day 60 in IgM-only memory B cells (CD27+/IgD−/IgM+) after TLR7 stimulation also predicted slower progression from CIS to MS. Phototherapy was without significant effect on B-cell IL-10 production. Conclusions: Reduced TNF responses after TLR7 stimulation in marginal zone-like B cells from participants administered phototherapy suggested treatment-associated priming effects that were detected upon subsequent polyclonal B-cell activation. Changes in responsiveness to TLR7 stimulation also suggested that IgM-only memory B cells may be important in conversion from CIS to MS.