Biosurfactants are attracting much interest due to their potential application as therapeutic agents in the medical and cosmetic field. Previous studies have demonstrated that biosurfactant such as sophorolipid (SL) exhibits immunomodulatory effects. In this article, we found the potential of sophorolipid for inhibiting histamine-induced itch and preliminarily explored its molecular basis. First, behavioral tests indicated that SL can remit the histamine-induced scratching behaviors of mice. Second, SL can suppress the the calcium influx induced by histamine, HTMT and VUF8430 in HaCaT cells. RT-PCR analysis showed that the histamine-induced upregulation of mRNA levels of phospholipase Cγ1, 1,4,5-trisphosphate receptor (IP3R), and protein kinase Cα can be inhibted by SL, suggesting that SL may impede the PLC/IP3R signaling pathway activated by histamine. In further tests, the capsaicin-induced calcium influx can also be inhibited by SL. The immunofluorescence and molecular docking analysis indicated that SL acts as an inhibitor of transient receptor potential vanilloid-1 (TRPV1) activation to decrease calcium influx against stimuli. In summary, these results revealed that SL may inhibit histamine-induced itch by decreasing PLC/IP3R signaling pathway activation and modulating TRPV1 activity. This paper indicates that SL can be a useful treatment for histamine-dependent itch.