Loquat (Eriobotrya japonica) is a model fruit for investigating flesh lignification during storage and response to chilling injury. However, the investigations of enzymes and coding genes and loquat fruit lignification under low-temperature storage are still limited. Here, the activity and transcript levels of up-stream enzymes of the phenylpropanoid pathway, including l-phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate:coenzyme A ligase (4CL), were investigated. The results indicated that activity of these enzymes was positively correlated with loquat fruit lignification and suppression of these increases by heat treatment (HT) and low-temperature conditioning (LTC) significantly alleviated loquat fruit lignification. Coding genes for these enzymes were subsequently isolated based on information from an RNA-seq database and expression of Ej4CL1 was found to be the most responsive to low temperature and inhibition by HT and LTC treatment, whereas the other genes were less responsive to these treatments. Furthermore, function of Ej4CL1 was analyzed by transient overexpression in tobacco leaves, where it stimulated lignin accumulation. Ej4CL1 may be a key candidate that involved in CI-related loquat fruit lignification.