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The analysis of the subcellular localization and function of dense granule proteins (GRAs) is of central importance for the understanding of host-parasite interaction and pathogenesis of Toxoplasma gondii infection. Here, we identified 15 novel GRAs and used C-terminal endogenous gene tagging to determine their localization at the intravacuolar network (IVN), parasitophorous vacuole (PV), or PV membrane (PVM) in the tachyzoites and at the periphery of the bradyzoites-containing cysts. The functions of the 15 gra genes were examined in type I RH strain and 5 of these gra genes were also evaluated in the cyst-forming type II Pru strain. The 15 novel gra genes were successfully disrupted by using CRISPR-Cas9 mediated homologous recombination and the results showed that 13 gra genes were not individually essential for T. gondii replication in vitro or virulence in mice during acute and chronic infection. Intriguingly, deletion of TGME49_266410 and TGME49_315910 in both RH and Pru strains decreased the parasite replication in vitro and attenuated its virulence, and also reduced the cyst-forming ability of the Pru strain in mice during chronic infection. Comparison of the transcriptomic profiles of the 15 gra genes suggests that they may play roles in other life cycle stages and genotypes of T. gondii. Taken together, our findings improve the understanding of T. gondii pathogenesis and demonstrate the involvement of two novel GRAs, TGME49_266410 and TGME49_315910, in the parasite replication and virulence. IMPORTANCE Dense granule proteins (GRAs) play important roles in Toxoplasma gondii pathogenicity. However, the functions of many putative GRAs have not been elucidated. Here, we found that 15 novel GRAs are secreted into intravacuolar network (IVN), parasitophorous vacuole (PV), or PV membrane (PVM) in tachyzoites and are located at the periphery of the bradyzoite-containing cysts. TGME49_266410 and TGME49_315910 were crucial to the growth of RH and Pru strains in vitro . Deletion of TGME49_266410 and TGME49_315910 attenuated the parasite virulence in mice. However, disruption of other 13 gra genes did not have a significant impact on the proliferation and pathogenicity of T. gondii in vitro or in vivo . The marked effects of the two novel GRAs (TGME49_266410 and TGME49_315910) on the in vitro growth and virulence of T. gondii are notable and warrant further elucidation of the temporal and spatial dynamics of translocation of these two novel GRAs and how do they interfere with host cell functions.