Src-family kinases (SFKs) are non-receptor-type tyrosine kinases, which have been shown to interact with a host of upstream regulators and downstream substrates that play important roles in regulating cell proliferation, migration, differentiation and adhesion. NLRP3 inflammasome is an important intracellular multi-molecular complex in the innate immune system that can control caspase-1 activity which in turn regulates maturation of the pro-inflammatory cytokine interleukin-1β (IL-1β) to against invaded microbial. Recently, SFKs have been revealed to participate in NLRP3 inflammasome activation by malaria hemozoin, asbestos or influenza A. In this study we used genetic knockout approach to further elucidate the roles and the molecular mechanisms of three SFKs (Lyn, Hck, Fgr) in regulating the NLRP3 inflammasome activation in murine macrophages. We found that Lyn, Hck and Fgr can regulate ATP- and Candida albicans-induced caspase-1 activation and IL-1β secretion in different manners. All kinases did not regulate LPS-elicited pro-IL-1β and NLRP3 expression, neither ATP-induced potassium efflux or channel permeability through P2X7 activation. We found that Lyn is a positive regulator for both stimuli-induced ROS production and caspase-1 activation, and promotes TXNIP interaction with NLRP3 under LPS/ATP treatment. Lyn does not alter the decrease of mitochondrial membrane potential induced by LPS/ATP and Candida. Furthermore Lyn also mediates C. albicans-induced cathepsin B activity. Nevertheless, Hck and Fgr are negative regulators for ATP- and Candida-induced IL-1β production. Notably both kinases unlike Lyn did not affect the increased ROS level caused by Candida. We also identified Lyn is an upstream signal molecule of Syk upon Candida infection, and Syk inhibition led to reduction of Candida responses in terms of IL-1β and ROS production. Furthermore, the ability of macrophages in phagocytosis of Candida was not altered by three SFKs, while intracellular pathogen killing ability of macrophages was attenuated in Lyn-/-, but enhanced in Hck-/- and Fgr-/- macrophages. Taken together the three major SFKs in murine macrophages, Lyn, Hck and Fgr, exert different actions in ATP- and Candida-elicited NLRP3 inflammasome activation. Positive regulation of ROS production contributes to the role of Lyn in inflammasome activation, while the exact regulating mechanisms of Hck and Fgr, which are negative regulators in this aspect, need further investigation. In addition, how SFKs differentially control invaded Candida in macrophages remains unclear and requires more study in the future.