C-type lectins (CTLs) are a class of proteins containing carbohydrate recognition domains (CRDs), which are characteristic modules that recognize various glycoconjugates and function primarily in immunity. CTLs have been reported to affect growth and development and positively regulate innate immunity inTribolium castaneum. However, the regulatory mechanisms of TcCTL16 proteins are still unclear. Here, spatiotemporal analyses displayed thatTcCTL16was highly expressed in late pupae and early adults.TcCTL16RNA interference in early larvae shortened their body length and narrowed their body width, leading to the death of 98% of the larvae in the pupal stage. Further analysis found that the expression level of muscle-regulation-related genes, includingcut,vestigial,erect wing,apterous, andspalt major, and muscle-composition-related genes, includingMyosin heavy chainandMyosin light chain, were obviously down-regulated afterTcCTL16silencing inT. castaneum. In addition, the transcription ofTcCTL16was mainly distributed in the hemolymph.TcCTL16was significantly upregulated after challenges with lipopolysaccharides, peptidoglycans,Escherichia coli, andStaphylococcus aureus. Recombinant CRDs of TcCTL16 bind directly to the tested bacteria (exceptBacillus subtilis); they also induce extensive bacterial agglutination in the presence of Ca2+. On the contrary, afterTcCTL16silencing in the late larval stage,T. castaneumwere able to develop normally. Moreover, the transcript levels of seven antimicrobial peptide genes (attacin2,defensins1,defensins2,coleoptericin1,coleoptericin2,cecropins2, andcecropins3) and one transcription factor gene (relish) were significantly increased underE. colichallenge and led to an increased survival rate ofT. castaneumwhen infected withS. aureusorE. coli, suggesting that TcCTL16 deficiency could be compensated for by increasing AMP expression via the IMD pathways inT. castaneum. In conclusion, this study found that TcCTL16 could be involved in developmental regulation in early larvae and compensate for the loss of CTL function by regulating the expression of AMPs in late larvae, thus laying a solid foundation for further studies onT. castaneumCTLs.