Profilin functions with formin in actin assembly, a process that regulates multiple aspects of plant development and immune responses. High-level eukaryotes contain multiple isoforms of profilin, formin, and actin, whose partner-specific interactions in actin assembly are not completely understood in plant development and defense responses. To examine the functionally distinct interactions between profilin and formin, we studied all five Arabidopsis profilins and their interactions with formin by using both in vitro biochemical and in vivo cell biology approaches. Unexpectedly, we found a previously undescribed negative regulatory function of AtPRF3 in AtFH1-mediated actin polymerization. The N-terminal 37 residues of AtPRF3 were identified to play a predominant role in inhibiting formin-mediated actin nucleation via their high affinity for the formin polyproline region and their triggering of the oligomerization of AtPRF3. Both in vivo and in vitro mechanistic studies of AtPRF3 revealed a universal mechanism in which the weak interaction between profilin and formin positively regulates actin assembly by ensuring rapid recycling of profilin, whereas profilin oligomerization negatively regulates actin polymerization. Upon recognition of the pathogen-associated molecular pattern, the gene transcription and protein degradation of AtPRF3 are modulated for actin assembly during plant innate immunity. The prf3 Arabidopsis plants show higher sensitivity to the bacterial flagellum peptide in both the plant growth and ROS responses. These findings demonstrate a profilin-mediated actin assembly mechanism underlying the plant immune responses.