Gametocidal (Gc) chromosomes can kill gametes that lack them by causing chromosomal breakage to ensure their preferential transmission. As an effective breeding tool, Gc chromosome has been used for creating chromosome translocation and deletion lines in wheat. This study preliminarily investigated the underlying molecular mechanism of long non-coding RNAs (lncRNAs) in Gc action. Here, DAPI and KI staining of the wheat pollen showed that CS-3C pollen fertility decreased. By sequencing transcriptome and lncRNAs libraries from the anthers of Triticum aestivum cv. Chinese Spring (CS) and the Chinese Spring-Gc 3C chromosome monosomic addition line (CS-3C), we identified 2,824 differentially expressed lncRNAs. Subsequently, functional annotation of the cis and trans-targets of DE-lncRNAs were conducted, and 798 targets corresponding to 516 DE-lncRNAs were identified which might be involved in anther and pollen development. As lncRNAs can serve as precursor and endogenous target mimics (eTM) of miRNA, we analyzed lncRNAs to miRBase with blast and found 110 differentially expressed lncRNAs as precursors for 28 miRNAs, and 36 DE-lncRNAs as potential eTMs for 14 miRNAs. Combined with our previous miRNA sequencing analysis, LTCONS_00117735 could specifically bind to tae-miR9657b-3p, thus affect the effect of tae-miR9657b-3p on target genes. We verified the effect of LTCONS_00117735 with miR9657 by transient transformation of tobacco leaves, and the results showed that LTCONS_00117735 could indeed bind to tae-miR9657b-3p. We screened LTCONS_00006278 which could interact with the target gene MTCNS_00006277 through cis interaction and analyzed its molecular biological function by rice genetic transformation. These results indicated that both lncRNA-directed regulation of mRNAs and miRNA expression could play a role in Gc action. Our study opened up a new avenue for uncovering the underlying molecular mechanism of Gc action.