Type VI secretion system (T6SS) is a highly conserved bacterial protein secretion system and is precisely regulated in Gram-negative pathogens. In Vibrio alginolyticus, an important fish pathogen, two complete T6SS gene clusters (T6SSVA1 and T6SSVA2) were identified. In this study, expression of a hemolysin coregulated protein (Hcp1), which is one of the hallmarks of T6SS, was found to be strictly regulated in this bacterium. We showed that the expression of Hcp1 was growth phase-dependent and the production of Hcp1 reached a maximum in the exponential phase. The expression of Hcp1 was positively and negatively regulated by quorum sensing regulators LuxO and LuxR, respectively. In addition, we observed that Hcp1 expression required the alternative sigma factor RpoN and the enhancer-binding protein VasH, which is encoded in T6SSVA1 gene cluster. Moreover, LuxR, RpoN, and VasH could positively regulate the expression of other T6SS genes. Taken together, we demonstrated that the expression of T6SS in V. alginolyticus was under the regulation of quorum sensing and alternative sigma factor.