Prosopis juliflora (mesquite) is largely used for feeding cattle and humans. Intoxication with the plant has been reported, mainly when it is the sole sustenance for cattle and goats, and is characterized by neuro-muscular alterations, spongiosis and gliosis. This study was conducted to investigate the direct effects of alkaloids extracted from P. juliflora leaves on astrocyte primary cultures. A total alkaloidal extract (TAE) was obtained using an acid/basic-modified extraction and was fractionated in silica gel column. The TAE and four cytotoxic fractions (F29/30, F31/33, F32 and F34/35), at concentrations ranging from 0.3 to 30 µg/mL, in the initial MTT scream tests, were tested for 24 h on rat cortical astrocytes primary cultures. A negative control was constituted by cells treated with the vehicle (0.1% DMSO). Morphological changes and glial cells activation were investigated through Rosenfeld’s staining and by immunocytochemistry for the protein OX-42, specific of activated microglia, and for the protein GFAP, a marker of reactive and mature astrocytes. To investigate if the alkaloids interfere on glial cells function, the production of nitric oxide (NO) was assessed by nitrite accumulation in the culture medium. We observed that the majority of astrocytes exposed to 3 mg/ml of TAE, F29/30 and F31/33 developed a stellate form presenting a dense and compact cell body with many processes strongly expressing GFAP. Treatment with 30 mg/ml TAE and fractions induced cytotoxicity characterized by a strong cell body contraction, very thin and long processes and condensed chromatin. The number of OX-42 positive microglial cells was also increased by 1,37 ± 0,2 to 4,1 ± 0,2 folds on the cultures treated with 3 mg/mL TAE and fractions. This effect was dose-dependant, and specially amplified when the cells were exposed to 30 mg/ml F32, the most cytotoxic fraction. Moreover, incubation with 3 mg/ml F32 or 30 mg/mL TAE, F29/30, F31/33 or F34/35 induced a significant accumulation of nitrite on the culture medium. Taken together these results show that the TAE and fractionated alkaloids from P. juliflora acts directly on glial cells inducing activation or cytotoxicity and stimulate NO production, and it may impact on neuronal damages observed on intoxicated animals.