Efficient human-to-human transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission are still not fully understood. In this study, we compared the respiratory droplet transmissibility of four H7N9 viruses that are genetic closely related and found that these viruses have dissimilar transmissibility in guinea pigs: A/Anhui/1/2013 (AH/1) transmitted efficiently, whereas the other three viruses did not transmit. The three non-transmissible viruses have one to eight amino acid differences compared with the AH/1 virus. To investigate which of these amino acids is important for transmission, we used reverse genetics to generate a series of reassortants and mutants in the AH/1 background and tested their transmissibility in guinea pigs. We found that the NA of the non-transmissible virus A/chicken/Shanghai/S1053/2013 had low enzymatic activity that impaired the transmission of AH/1 virus, and three amino acid mutations--V292I and K627E in PB2 and D156E in M1--independently abolished the transmission of the AH/1 virus. We further found that an NA reassortant and three single-amino-acid mutants replicated less efficiently than the AH/1 virus in A549 cells, and that the amino acid at position 156 of M1 affected the morphology of H7N9 viruses. Our study identifies key amino acids in PB2 and M1 that play important roles in H7N9 influenza virus transmission and provides new insights into the transmissibility of influenza virus.Importance Efficient transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission remain poorly understood. H7N9 influenza viruses, which emerged in 2013 in China, have caused over 1,560 human infection cases, showing clear pandemic potential. Previous studies have shown that the H7N9 viruses differ in their transmissibility in animal models. Here we found two amino acids in PB2 (292V and 627K) and one in M1 (156D) that are extremely important for H7N9 virus transmission. Of note, PB2 292V and M1 156D appear in most H7N9 viruses, and the PB2 627K mutation could easily occur when the H7N9 virus replicates in humans. Our study thus identifies new amino acids that are important for influenza virus transmission and suggests that just a few key amino acid changes can render the H7N9 virus transmissible in mammals.