The effects of Δ⁹-tetrahydricannabinol (Δ⁹-THC), cannabidiol (CBD), cannabidiolic acid (CBDA), and 2-methyl-2’-fluoro-anandamide (MF-AEA, a stable analog of anandamide) on the enzymatic activity of purified human calpain-1 (CAPN1) were investigated in the present study. Although leupeptin, a calpain inhibitor, reduced calpain-1 activity in a dose-dependent manner (1, 5, and 25 μM), among the four cannabinoids tested, Δ⁹-THC and CBD exerted stimulatory effects on calpain-1 enzymatic activity in the same concentration range as leupeptin. CBDA and MF-AEA did not modulate calpain-1 activity, indicating that the phenol structure in Δ⁹-THC/CBD is a key point, and the carboxylic acid moiety appears to be negatively involved. This is the first study to show that the phytocannabinoids, Δ⁹-THC and CBD have the ability to activate the enzymatic activity of human calpain-1.