Objective To investigate the production and removal of cyclobutane pyrimidine dimer (CPD) by HaCaT cells after UVB irradiation, and the effect of baicalin in this process. Methods HaCaT cells were cultured and irradiated with given dosages of UVB, and the production and removal of CPD by HaCaT cells at given time points after UVB irradiation were assessed by immunohistochemical method. In parallel studies, HaCaT cells were preincubated with baicalin, and the effect on CPD was evaluated. Results The damage to HaCaT cells was dependent on the dosage of UVB radiation. After irradiation with 30 mJ/cm2 of UVB, CPD formation peaked at 0.5 h. CPD was removed rapidly from HaCaT cells during the first 4 h; the rate of removal decreased thereafter, and the removal was almost complete by 24 h after the irradiation. The amount of CPD decreased significantly in HaCaT cells that were preincubated with baicalin solution before UVB irradiation than that in those without the preincubation (U = 2.324, P < 0.05). Conclusions UVB radiation results in a dose dependent damage to HaCaT cells. There are two phases in the removal of CPD after UVB irradiation: a rapid phase and a slow phase. Pretreatment of HaCat cells with baicalin before UVB irradiation can decrease the production of CPD.