The present study aimed to determine phospholipase C (PLC) activation status during changes promoted by Azospirillum brasilense Sp245 in wheat seedlings. Germinated Triticum aestivum seeds by 3 days were inoculated with different A. brasilense concentrations (1 × 10⁶ and 4 × 10⁶ CFU/mL) and PLC enzyme activity assayed at different times. Root and leaf length, as well as total fresh weight were assessed as growth parameters; moreover, changes in root morphology were analyzed. PLC activity was measured by molybdate assay. Neomycin and LaCI₃ treatments verified PLC- and/or Ca²⁺ -dependent effects on inoculated wheat, respectively. A. brasilense increased PLC activity 15–30 min after inoculation. Ca²⁺ -channel blocker LaCI₃ decreased PLC activity, and activity did not recover with A. brasilense. The A. brasilense mutant FAJ009, impaired in auxin production, showed decreased PLC activity versus wild type. PLC activity was inhibited by neomycin (PLC inhibitor), concomitant with a decrease in total fresh weight. Exogenous addition of diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3), secondary messengers produced by the activity of PLC, increased root hair length. The putative role of PLC enzyme activity in morphological changes promoted by A. brasilense in wheat seedlings is discussed.