Silkworm is used as a model organism to analyze two standard complex traits, which are high and low silk yields. To understand the molecular mechanisms of silk production, the posterior silk glands aged to the third day of the fifth instar were analyzed from the ZB strain with low silk production and from the control strain Lan10. Using isobaric tags for relative and absolute quantification (iTRAQ) quantitative shotgun proteomics and RNA-sequencing-based transcriptomics, 139 proteins and 630 transcripts were identified as novel in the ZB strain compared with the Lan10 strain, indicating that these results significantly expand the coverage of proteins and transcripts of the posterior silk glands in the silkworm. Of the 89 differently changed proteins, 23 were increased, and 66 were decreased. Of the 788 transcripts, 779 were upregulated, and 9 were downregulated. These results confirm that decreased energy utilization/protein translation and enhanced protein degradation are the key factors in lower silk production. Moreover, this study provides novel insight into the molecular changes that may result in lower silk production, namely, a combination of impaired transcription activity, missed protein folding/transport, and lowered yields of the main components of fibroin, along with weakened growth/development of the posterior silk gland.