Crotamiton is an anti-scabies drug, but it was recently found that crotamiton also suppresses non-scabietic itching in mice. However, the underlying mechanism is largely unclear. Therefore, aim of the study is to investigate mechanisms of the anti-pruritic effect of crotamiton for non-scabietic itching. Histamine and chloroquine are used as non-scabietic pruritogens. The effect of crotamiton was identified using fluorometric intracellular calcium assays in HEK293T cells and primary cultured dorsal root ganglion (DRG) neurons. Further in vivo effect was evaluated by scratching behavior tests. Crotamiton strongly inhibited histamine-induced calcium influx in HEK293T cells, expressing both histamine receptor 1 (H1R) and transient receptor potential vanilloid 1 (TRPV1), as a model of histamine-induced itching. Similarly, it also blocked chloroquine-induced calcium influx in HEK293T cells, expressing both Mas-related G-protein-coupled receptor A3 (MRGPRA3) and transient receptor potential A1 (TRPA1), as a model of histamine-independent itching. Furthermore, crotamiton also suppressed both histamine- and chloroquine-induced calcium influx in primary cultures of mouse DRG. Additionally, crotamiton strongly suppressed histamine- and chloroquine-induced scratching in mice. Overall, it was found that crotamiton has an anti-pruritic effect against non-scabietic itching by histamine and chloroquine. Therefore, crotamiton may be used as a general anti-pruritic agent, irrespective of the presence of scabies.